DNA encoding galanin GALR3 receptors and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid encoding a mammalian galanin receptor, an isolated galanin receptor protein, vectors comprising isolated nucleic acid encoding a mammalian galanin receptor, cells comprising such vectors, antibodies directed to a mammalian galanin receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian galanin receptor, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding a mammalian galanin receptor, nonhuman transgenic animals which express DNA encoding a normal or a mutant mammalian galanin receptor, as well as methods of determining binding of compounds to mammalian galanin receptors.

This application is a continuation-in-part of U.S. Ser. No. 08/787,261,filed Jan. 24, 1997, now abandoned, which is a continuation-in-part ofU.S. Ser. No. 08/767,964, filed Dec. 17, 1996, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/728,139, filed Oct. 9, 1996,now abandoned, the contents of which are incorporated by reference.Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the sequence listing and the claims.

BACKGROUND OF THE INVENTION

The neuropeptide galanin and its receptors hold great promise as targetsfor the development of novel therapeutic agents. Galanin is widelydistributed throughout the peripheral and central nervous systems and isassociated with the regulation of processes such as somatosensorytransmission, smooth muscle contractility, hormone release, and feeding(for review, see Bartfai et al., 1993). In the periphery galanin isfound in the adrenal medulla, uterus, gastrointestinal tract, dorsalroot ganglia (DRG), and sympathetic neurons. Galanin released fromsympathetic nerve terminals in the pancreas is a potent regulator ofinsulin release in several species (Ahrén and Lindskog, 1992; Boyle etal., 1994), suggesting a potential role for galanin in the etiology ortreatment of diabetes. High levels of galanin are observed in human andrat anterior pituitary where galanin mRNA levels are potentlyupregulated by estrogen (Vrontakis et al., 1987; Kaplan et al., 1988).The presence of galanin in the hypothalamic-pituitary-adrenal axiscoupled with its potent hormonal effects has led to the suggestion thatgalanin may play an integral role in the hormonal response to stress(Bartfai et al., 1993).

Within the CNS galanin-containing cell bodies are found in thehypothalamus, hippocampus, amygdala, basal forebrain, brainstem nuclei,and spinal cord, with highest concentrations of galanin in thehypothalamus and pituitary (Skofitsch and Jacobowitz, 1985; Bennet etal., 1991; Merchenthaler et al., 1993). The distribution of galaninreceptors in the CNS generally complements that of galanin peptide, withhigh levels of galanin binding observed in the hypothalamus, amygdala,hippocampus, brainstem and dorsal spinal cord (Skofitsch et al., 1986;Merchenthaler et al., 1993; see Bartfai et al., 1993). Accordingly,agents modulating the activity of galanin receptors would have multiplepotential therapeutic applications in the CNS. One of the most importantof these is the regulation of food intake. Galanin injected into theparaventricular nucleus (PVN) of the hypothalamus stimulates feeding insatiated rats (Kyrkouli et al., 1990), an effect which is blocked by thepeptide galanin antagonist M40 (Crawley et al., 1993). In freely feedingrats, PVN injection of galanin preferentially stimulates fat-preferringfeeding (Tempel et al., 1988); importantly, the galanin antagonist M40administered alone decreases overall fat intake (Leibowitz and Kim,1992). These data indicate that specific receptors in the hypothalamusmediate the effects of galanin on feeding behavior, and further suggestthat agents acting at hypothalamic galanin receptors may betherapeutically useful in the treatment of human eating disorders.

Galanin receptors elsewhere in the CNS may also serve as therapeutictargets. In the spinal cord galanin is released from the terminals ofsensory neurons as well as spinal interneurons and appears to play arole in the regulation of pain threshold (Wiesenfeld-Hallin et al.,1992). Intrathecal galanin potentiates the anti-nociceptive effects ofmorphine in rats and produces analgesia when administered alone(Wiesenfeld-Hallin et al., 1993; Post et al., 1988); galanin receptoragonists may therefore be useful as analgesic agents in the spinal cord.Galanin may also play a role in the development of Alzheimer's disease.In the hippocampus galanin inhibits both the release (Fisone et al.,1987) and efficacy (Palazzi et al., 1988) of acetylcholine, causing animpairment of cognitive functions (Sundström et al., 1988). Autopsysamples from humans afflicted with Alzheimer's disease reveal agalaninergic hyperinnervation of the nucleus basalis (Chan-Palay, 1988),suggesting a role for galanin in the impaired cognition characterizingAlzheimer's disease. Together these data suggest that a galaninantagonist may be effective in ameliorating the symptoms of Alzheimer'sdisease (see Crawley, 1993). This hypothesis is supported by the reportthat intraventricular administration of the peptide galanin antagonistM35 improves cognitive performance in rats (Ögren et al., 1992). Humangalanin receptors thus provide targets for therapeutic intervention inmultiple CNS disorders.

High-affinity galanin binding sites have been characterized in brain,spinal cord, pancreatic islets and cell lines, and gastrointestinalsmooth muscle in several mammalian species, and all show similaraffinity for ¹²⁵I-porcine galanin (˜0.5-1 nM). Nevertheless, recent invitro and in vivo pharmacological studies in which fragments andanalogues of galanin were used suggest the existence of multiple galaninreceptor subtypes. For example, a galanin binding site in guinea pigstomach has been reported that exhibits high affinity for porcinegalanin (3-29) (Gu, et al. 1995), which is inactive at CNS galaninreceptors. The chimeric galanin analogue M15 (galantide) acts asantagonist at CNS galanin receptors (Bartfai et al., 1991) but as a fullagonist in gastrointestinal smooth muscle (Gu et al., 1993). Similarly,the galanin-receptor ligand M40 acts as a weak agonist in RINm5Finsulinoma cells and a full antagonist in brain (Bartfai et al, 1993a).The pharmacological profile of galanin receptors in RINm5F cells can befurther distinguished from those in brain by the differential affinitiesof [D-Tyr²]- and [D-Phe²]-galanin analogues (Lagny-Pourmir et al.,1989). The chimeric galanin analogue M35 displaces ¹²⁵I-galanin bindingto RINm5F membranes in a biphasic manner, suggesting the presence ofmultiple galanin receptor subtypes, in this cell line (Gregersen et al.,1993).

Multiple galanin receptor subtypes may also co-exist within the CNS.Galanin receptors in the dorsal hippocampus exhibit high affinity forGal (1-15) but not for Gal (1-29) (Hedlund et al., 1992), suggestingthat endogenous proteolytic processing may release bioactive fragmentsof galanin to act at distinct receptors. The rat pituitary exhibitshigh-affinity binding for 12Sj-Bolton and Hunter (N-terminus) -labeledgalanin (1-29) but not for [¹²⁵I] Tyr²⁶-porcine galanin (Wynick et al.,1993), suggesting that the pituitary galanin receptor is aC-terminus-preferring subtype. Spinal cord galanin binding sites, whilesimilar to those in brain, show an affinity for the chimeric peptideantagonist M35 intermediate between the brain and smooth muscle (Bartfaiet al., 1991), raising the possibility of further heterogeneity.

A galanin receptor cDNA was recently isolated by expression cloning froma human Bowes melanoma cell line (Habert-Ortoli et al., 1994). Thepharmacological profile exhibited by this receptor is similar to thatobserved in brain and pancreas, and on that basis the receptor has beentermed GALR1. The cloned human GALR1 receptor (“hGALR1”) binds nativehuman, porcine and rat galanin with ˜1 nM affinity (K_(i) vs.¹²⁵I-galanin) and porcine galanin 1-16 at a slightly lower affinity (˜5nM). Porcine galanin 3-29 does not bind to the receptor. The GALR1receptor appears to couple to inhibition of adenylate cyclase, withhalf-maximal inhibition of forskolin-stimulated cAMP production by 1 nMgalanin, and maximal inhibition occurring at about 1 μM.

Recently the rat homologue of GALRl (“rGALR1”) was cloned from theRIN14B pancreatic cell line (Burgevin, et al., (1995), Parker et al.,1995. The pharmacologic data reported to date do not suggest substantialdifferences between the pharmacologic properties of the rat and humanGALR1 receptors. Localization studies reveal GALR1 mRNA in rathypothalamus, ventral hippocampus, brainstem, and spinal cord (Gustafsonet al., 1996), regions consistent with roles for galanin in feeding,cognition, and pain transmission. However, GALR1 appears to be distinctfrom the pituitary and hippocampal receptor subtypes described above.

The indication of multiple galanin receptor subtypes within the brainunderscores the importance of defining galanin receptor heterogeneity atthe molecular level in order to develop specific therapeutic agents forCNS disorders. Pharmacological tools capable of distinguishing galaninreceptor subtypes in tissue preparations are only beginning to appear.Several high-affinity peptide-based galanin antagonists have beendeveloped and are proving useful in probing the functions of galaninreceptors (see Bartfai et al., 1993), but their peptide characterprecludes practical use as therapeutic agents. In light of galanin'smultiple neuroendocrine roles, therapeutic agents targeting a specificdisorder must be selective for the appropriate receptor subtype tominimize side effects.

Accordingly, applicants have endeavored to clone the entire family ofgalanin receptors for use in target-based drug design programs. Theidentification of non-peptide agents acting selectively only at specificgalanin receptors will be greatly facilitated by the cloning,expression, and characterization of the galanin receptor family.

Applicants have recently isolated by expression cloning from a rathypothalamic cDNA library a novel galanin receptor, termed “GALR2,” notdescribed herein, which is distinguishable from GALR1 both by its uniquesequence and distinct pharmacologic properties.

Applicants now report the isolation of a novel galanin receptor subtype,referred to herein as “GALR3,” from a rat hypothalamic cDNA library.This discovery provides a novel approach, through the use ofheterologous expression systems, to develop subtype selective,high-affinity non-peptide compounds that could serve as therapeuticagents for eating disorders, diabetes, pain, depression, ischemia, andAlzheimer's disease. Pathophysiological disorders proposed to be linkedto galanin receptor activation include eating disorders, diabetes, pain,depression, ischemia, Alzheimer's disease and reproductive disorders.Accordingly, treatment of such disorders may be effected by theadministration of GALR3 receptor-selective compounds. The presence ofgalanin binding sites in multiple CNS areas suggests that GALR3receptors may also play a role in cognition, analgesia, sensoryprocessing (olfactory, visual), processing of visceral information,motor coordination, modulation of dopaminergic activity, neuroendocrinefunction, sleep disorders, migraine, and anxiety.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a GALR3galanin receptor. This invention also provides an isolated GALR3receptor protein. This invention also provides a purified GALR3 receptorprotein . This invention further provides DNA, cDNA, genomic DNA, RNA,and mRNA encoding the GALR3 receptor.

This invention further provides a vector comprising the GALR3 receptor.Such a vector may be adapted for expression of the GALR3 receptor inmammalian or non-mammalian cells. This invention also provides a plasmidwhich comprises the regulatory elements necessary for expression ofGALR3 nucleic acid in a mammalian cell operatively linked to a nucleicacid encoding the GALR3 receptor so as to permit expression thereof,designated K1086 (ATCC Accession No. 97747). This invention alsoprovides a plasmid which comprises the regulatory elements necessary forexpression of GALR3 nucleic acid in a mammalian cell operatively linkedto a nucleic acid encoding a human GALR3 receptor so as to permitexpression thereof, designated pEXJ-hGalR3 (ATCC Accession No. 97827).This invention provides mammalian cells comprising the above-describedplasmid or vector. This invention also provides a membrane preparationisolated from the cells.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR3 receptor contained in plasmid K1086.This invention still further provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequencedescribed in FIG. 1 (Seq. ID No. 1) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 1 (Seq. ID No. 1).

In yet another embodiment, the GALR3 receptor is the rat GALR3 receptorhaving substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2. In another embodiment, the GALR3 receptor isthe rat GALR3 receptor having the amino acid sequence shown in FIG. 2.In another embodiment, the GALR3 receptor is the human GALR3 receptor.In another embodiment, the GALR3 receptor is the human GALR3 receptorencoded by the coding sequence of plasmid pEXJ-hGalR3. This inventionalso provides a nucleic acid probe comprising at least 15 nucleotides,which probe specifically hybridizes with a nucleic acid encoding a GALR3receptor, wherein the probe has a unique sequence corresponding to asequence present within one of the two strands of the nucleic acidencoding the GALR3 receptor contained in plasmid pEXJ-hGalR3. Thisinvention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequencedescribed in FIG. 3 (Seq. ID No. 3) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 3 (Seq. ID No. 3).

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a GALR3 galaninreceptor, so as to prevent translation of the mRNA. This invention alsoprovides an antisense oligonucleotide having a sequence capable ofspecifically hybridizing to the genomic DNA molecule encoding a GALR3receptor.

This invention provides an antibody directed to a GALR3 receptor. Thisinvention also provides a monoclonal antibody directed to an epitope ofa GALR3 receptor, which epitope is present on the surface of a cellexpressing a GALR3 receptor.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a GALR3receptor by passing through a cell membrane and binding specificallywith mRNA encoding a GALR3 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In an embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

This invention provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a GALR3receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a GALR3 receptorand a pharmaceutically acceptable carrier.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a GALR3 receptor. This invention provides a transgenic nonhumanmammal comprising a homologous recombination knockout of the nativeGALR3 receptor. This invention provides a transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding aGALR3 receptor so placed as to be transcribed into antisense mRNA whichis complementary to mRNA encoding a GALR3 receptor and which hybridizesto MRNA encoding a GALR3 receptor thereby reducing its translation.

This invention also provides a process for determining whether acompound can specifically bind to a GALR3 receptor which comprisescontacting a cell transfected with and expressing DNA encoding the GALR3receptor with the compound under conditions permitting binding ofcompounds to such receptor, and detecting the presence of any suchcompound specifically bound to the GALR3 receptor, so as to therebydetermine whether the ligand specifically binds to the GALR3 receptor.

This invention provides a process for determining whether a compound canspecifically bind to a GALR3 receptor which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR3 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting binding of compounds to such receptor, and detecting t hepresence of the compound specifically bound to the GALR3 receptor, so asto thereby determine whether the compound specifically binds to theGALR3 receptor.

In one embodiment, the GALR3 receptor is a mammalian GALR3 receptor. Inanother embodiment, the GALR3 receptor is a rat GALR3 receptor. In stillanother embodiment, the GALR3 receptor has substantially the same aminoacid sequence encoded by the plasmid K1086. In a still furtherembodiment, the GALR3 receptor has the amino acid sequence encoded bythe plasmid K1086. In another embodiment, the GALR3 receptor is a humanGALR3 receptor.

This invention provides a process for determining whether a compound isa GALR3 receptor agonist which comprises contacting a cell transfectedwith and expressing DNA encoding the GALR3 receptor with the compoundunder conditions permitting the activation of the GALR3 receptor, anddetecting an increase in GALR3 receptor activity, so as to therebydetermine whether the compound is a GALR3 receptor agonist.

This invention provides a process for determining whether a compound isa GALR3 receptor antagonist which comprises contacting a celltransfected with and expressing DNA encoding the GALR3 receptor with thecompound in the presence of a known GALR3 receptor agonist, such asgalanin, under conditions permitting the activation of the GALR3receptor, and detecting a decrease in GALR3 receptor activity, so as tothereby determine whether the compound is a GALR3 receptor antagonist.

This invention provides a compound determined by the above-describedprocesses. In one embodiment of the above-described processes, thecompound is not previously known. In another embodiment, the compound isnot known to bind a GALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR3 receptor to identify a compoundwhich specifically binds to the GALR3 receptor, which comprises (a)contacting cells transfected with and expressing DNA encoding the GALR3receptor with a compound known to bind specifically to the GALR3receptor; (b) contacting the preparation of step (a) with the pluralityof compounds not known to bind specifically to the GALR3 receptor, underconditions permitting binding of compounds known to bind the GALR3receptor; (c) determining whether the binding of the compound known tobind to the GALR3 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to theGALR3 receptor of each compound included in the plurality of compounds,so as to thereby identify the compound which specifically binds to theGALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a GALR3 receptor to identify a compoundwhich activates the GALR3 receptor which comprises (a) contacting cellstransfected with and expressing the GALR3 receptor with the plurality ofcompounds not known to activate the GALR3 receptor, under conditionspermitting activation of the GALR3 receptor; (b) determining whether theactivity of the GALR3 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the GALR3 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the GALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a GALR3 receptor toidentify a compound which inhibits the activation of the GALR3 receptor,which comprises (a) preparing a cell extract from cells transfected withand expressing DNA encoding the GALR3 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe plurality of compounds in the presence of a known GALR3 receptoragonist, under conditions permitting activation of the GALR3 receptor;(b) determining whether the activation of the GALR3 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the GALR3 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the GALR3 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the GALR3 receptor.

This invention provides a method of detecting expression of a GALR3receptor by detecting the presence of mRNA coding for the GALR3 receptorwhich comprises obtaining total mRNA from the cell and contacting themRNA so obtained with the above-described nucleic acid probe underhybridizing conditions, detecting the presence of mRNA hybridized to theprobe, and thereby detecting the expression of the GALR3 receptor by thecell.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of aGALR3 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective todecrease the activity of the GALR3 receptor in the subject, therebytreating the abnormality in the subject. In an embodiment, theabnormality is obesity. In another embodiment, the abnormality isbulimia.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a GALR3receptor which comprises administering to a subject an effective amountof the above-described pharmaceutical composition effective to activatethe GALR3 receptor in the subject. In an embodiment, the abnormalcondition is anorexia.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific human GALR3 receptorallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human GALR3 receptor and labeled with a detectable marker;(e) detecting labeled bands which have hybridized to DNA encoding ahuman GALR3 receptor labeled with a detectable marker to create a uniqueband pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps a-e; and (g)comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

This invention provides a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In an embodiment, thecompound is a GALR3 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In another embodimentthe compound is administered in combination with food.

In yet another embodiment the compound is a GALR3 receptor agonist andthe amount is effective to increase the consumption of food by thesubject. In a still further embodiment, the compound is administered incombination with food. In other embodiments the subject is a vertebrate,a mammal, a human or a canine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Nucleotide coding sequence of the rat hypothalamic galanin GALR3receptor (Seq. I.D. No. 1), with partial 5′ and 3′ untranslatedsequences. Start and stop codons are underlined.

FIG. 2 Deduced amino acid sequence of the rat hypothalamic galanin GALR3receptor (Seq. I.D. No. 2) encoded by the rat nucleotide sequence shownin FIG. 1.

FIG. 3 Nucleotide coding sequence of the human galanin GALR3 receptor(Seq. I.D. No. 3), with partial 5′ and 3′ untranslated sequences. Startand stop codons are underlined.

FIG. 4 Deduced amino acid sequence of the human galanin GALR3 receptor(Seq. I.D. No. 4) encoded by the human nucleotide sequence shown in FIG.3. The nucleotide sequence shown in FIG. 3 is translated from nucleotide1 to the stop codon. Two possible starting methionines are underlined.

FIGS. 5A-5D Amino acid sequence alignment of the rat GALR3 receptor (toprow) (Seq. ID No. 2), human GALR3 receptor (middle row) (Seq. ID No. 4)and rat GALR1 receptor (bottom row) (Seq. ID No. 5). Transmembranedomains (TM 1-7) are indicated by brackets above the sequence.

FIGS. 6A-6B FIG. 6A: Long continuous trace (3 segments) demonstratesgalanin responsivity and sensitivity to Ba⁺⁺ block in an oocyteexpressing hGalR3 and GIRK1 and GIRK4. Switching from ND96 to 1/2 hKsolution causes the appearance of a large resting (inward) K⁺ currentthat increases further upon transient addition of 3 μM galanin.Subsequent addition of 300 μM Ba⁺⁺ largely blocks both the resting andgalanin-stimulated K⁺ currents. After removal of Ba⁺⁺ galaninresponsivity is partially restored. FIG. 6B: Concentration-responsecharacteristic of a second oocyte expressing both hGalR3 and GIRKs.Stepwise increases in the concentration of porcine galanin from 10 to10,000 nM result in a saturable increase in inward current.

FIG. 7 Pertussis toxin sensitivity of GalR3 and GalR1 stimulation ofGIRK currents. Normalized mean currents elicited by 0.1 μM (GalR1) and 1μM (GalR3) galanin in oocytes injected 3 h prior with 2 ng of pertussistoxin compared to water-injected oocytes. For oocytes expressing GalR2and α1a receptors, the response amplitude was measured as the peak ofthe Cl⁻ current stimulated by 1 μM galanin or epinephrine, respectively.Number of observations appears in parenthesis below the x-axis. Apparentabsence of a bar indicates an amplitude of 0 (no response abovebaseline).

FIGS. 8A-8F Concentration-response relations for 6 peptides at GalR3receptors expressed in oocytes. FIG. 8A: M32; FIG. 8B: porcine galanin;FIG. 8C: C7; FIG. 8D: Gal −7-29; FIG. 8E: Gal 1-16; FIG. 8F: M40.Measurements of GIRK currents were made as shown for galanin in FIG. 6B.For all peptides, responses from 3-6 oocytes were averaged for each datapoint. Curves were fitted with the logistic equationI=Imax/(1+(EC₅₀/[Agonist])^(n)), where EC₅₀ is the concentration ofagonist that produced half-maximal activation, and n the Hillcoefficient. Fits were made with a Marguardt-Levenberg non-linearleast-squares curve fitting algorithm.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

C = cytosine A = adenine T = thymine G = guanine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the receptors of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thereceptors of the subject invention.

The activity of a G-protein coupled receptor such as a galanin receptormay be measured using any of a variety of functional assays which arewell-known in the art, in which activation of the receptor in questionresults in an observable change in the level of some second messenger,including but not limited to adenylate cyclase, calcium mobilization,arachidonic acid release, ion channel activity, inositol phospholipidhydrolysis or guanylyl cyclase. Heterologous expression systemsutilizing appropriate host cells to express the nucleic acid of thesubject invention are used to obtain the desired second messengercoupling. Receptor activity may also be assayed in an oocyte expressionsystem, using methods well known in the art.

This invention provides an isolated nucleic acid encoding a GALR3galanin receptor. This invention further provides a recombinant nucleicacid encoding a GALR3 galanin receptor. In an embodiment, the galaninreceptor is a vertebrate or a mammalian GALR3 receptor. In anotherembodiment, the galanin receptor is a rat GALR3 receptor. In anotherembodiment, the galanin receptor is a human GALR3 receptor. In anembodiment, the isolated nucleic acid encodes a receptor characterizedby an amino acid sequence in the transmembrane region, which has ahomology of 70% or higher to the amino acid sequence in thetransmembrane region of the rat galanin GALR3 receptor and a homology ofless than 70% to the amino acid sequence in the transmembrane region ofany GALR1 receptor. In an embodiment, the GALR3 receptor is a rat GALR3receptor. In another embodiment, the GALR3 receptor is a human GALR3receptor.

This invention provides an isolated nucleic acid encoding a GALR3receptor having the same or substantially the A same amino acid sequenceas the amino acid sequence encoded by the plasmid K1086 (ATCC AccessionNo. 97747). In an embodiment, the nucleic acid is DNA. This inventionfurther provides an isolated nucleic acid encoding a rat GALR3 receptorhaving the amino acid sequence encoded by the plasmid K1086. Thisinvention provides an isolated nucleic acid encoding a GALR3 receptorhaving substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 (Seq. I.D. No. 2). In another embodiment, theGALR3 receptor is the rat GALR3 receptor having the amino acid sequenceshown in FIG. 2 (Seq. ID NO. 2). In another embodiment, the nucleic acidcomprises at least an intron. In still another embodiment, the nucleicacid comprises alternately spliced nucleic acid transcribed from thenucleic acid contained in plasmid K1086. In an embodiment, thealternately spliced nucleic acid is mRNA transcribed from DNA encoding agalanin receptor.

In an embodiment, the GALR3 receptor is a human GALR3 receptor. Thisinvention provides an isolated nucleic acid encoding a human GALR3receptor having the same or substantially the same amino acid sequenceas the amino acid sequence encoded by plasmid pEXJ-hGalR3 (ATCCAccession No. 97827). This invention provides an isolated nucleic acidencoding a human GALR3 receptor, wherein the human GALR3 receptor has asequence, which sequence comprises substantially the same amino acidsequence as the sequence shown in FIG. 4 (Seq. I.D. No. 4) from aminoacid 60 through amino acid 427. In another embodiment, the GALR3receptor has a sequence, which sequence comprises the sequence shown inFIG. 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.

In another embodiment, the nucleic acid encoding the human GALR3receptor comprises an intron. In still another embodiment, the nucleicacid encoding the human GALR3 receptor comprises alternately splicednucleic acid.

The fact that introns are found in many G protein coupled receptorsraises the possibility that introns could exist in coding or non-codingregions of GALR3; if so, a spliced form of mRNA may encode additionalamino acids either upstream of the currently defined starting methionineor within the coding region. Further, the existence and use ofalternative exons is possible, whereby the mRNA may encode differentamino acids within the region comprising the exon. In addition, singleamino acid substitutions may arise via the mechanism of RNA editing suchthat the amino acid sequence of the expressed protein is different thanthat encoded by the original gene (Burns et al., 1996; Chu et al.,1996). Such variants may exhibit pharmacologic properties differing fromthe receptor encoded by the original gene.

This invention provides a splice variant of the GALR3 receptorsdisclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding rat and human GALR3 receptors.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is DNA. In one embodiment, the DNA is cDNA. Inanother embodiment, the DNA is genomic DNA. In still another embodiment,the nucleic acid molecule is RNA. Methods for production andmanipulation of nucleic acid molecules are well known in the art.

This invention provides a vector encoding the nucleic acid of humanGALR3 receptor.

In another embodiment, the nucleic acid encodes a vertebrate GALR3receptor. In a separate embodiment, the nucleic acid encodes a mammalianGALR3 receptor. In another embodiment, the nucleic acid encodes a ratGALR3 receptor. In still another embodiment, the nucleic acid encodes ahuman GALR3 receptor.

This invention further provides nucleic acid which is degenerate withrespect to the DNA comprising the coding sequence of the plasmid K1086(ATCC Accession No. 97747). This invention further provides nucleic acidwhich is degenerate with respect to any DNA encoding a GALR3 receptor.In an embodiment, the nucleic acid comprises a nucleotide sequence whichis degenerate with respect to the nucleotide sequence of plasmid K1086,that is, a nucleotide sequence which is translated into the same aminoacid sequence. In an embodiment, the nucleic acid comprises a nucleotidesequence which is degenerate with respect to the nucleotide sequence ofplasmid pEXJ-rGalR3T (ATCC Accession No. 97826). In another embodiment,the nucleic acid comprises a nucleotide sequence which is degeneratewith respect to the nucleotide sequence of plasmid pEXJ-hGalR3 (ATCCAccession No. 97827).

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the GALR3 galanin receptor, butwhich should not produce phenotypic changes. Alternatively, thisinvention also encompasses DNAs, cDNAs, and RNAs which hybridize to theDNA, cDNA, and RNA of the subject invention. Hybridization methods arewell known to those of skill in the art.

The nucleic acids of the subject invention also include nucleic acidmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

G-protein coupled receptors such as the GALR3 receptors of the presentinvention are characterized by the ability of an agonist to promote theformation of a high-affinity ternary complex between the agonist, thereceptor, and an intracellular G-protein. This complex is formed in thepresence of physiological concentrations of GTP, and results in thedissociation of the alpha subunit of the G protein from the beta andgamma subunits of the G protein, which further results in a functionalresponse, i.e., activation of downstream effectors such as adenylylcyclase or phospholipase C. This high-affinity complex is transient evenin the presence of GTP, so that if the complex is destablized, theaffinity of the receptor for agonists is reduced. Thus, if a receptor isnot optimally coupled to G protein under the conditions of an assay, anagonist will bind to the receptor with low affinity. In contrast, theaffinity of the receptor for an antagonist is normally not significantlyaffected by the presence or absence of G protein. Functional assays maybe used to determine whether a compound binds to the receptor, but maybe more time-consuming or difficult to perform than a binding assay.Therefore, it may desirable to produce a receptor which will bind toagonists with high affinity in a binding assay. Examples of modifiedreceptors which bind agonists with high affinity are disclosed in WO96/14331, which describes neuropeptide Y receptors modified in the thirdintracellular domain. The modifications may include deletions of 6-13amino acids in the third intracellular loop. Such deletions preferablyend immediately before the polar or charged residue at the beginning ofhelix six. In an embodiment, the deleted amino acids are at the carboxyterminus of the third intracellular domain. Such modified receptors maybe produced using methods well-known in the art such as site-directedmutagenesis or recombinant techniques using restriction enzymes.

This invention provides an isolated nucleic acid encoding a modifiedGALR3 receptor, which differs from a GALR3 receptor by having an aminoacid(s) deletion, replacement or addition in the third intracellulardomain. In one embodiment, the modified GALR3 receptor differs by havinga deletion in the third intracellular domain. In another embodiment, themodified GALR3 receptor differs by having an amino acid replacement oraddition to the third intracellular domain.

The modified receptors of this invention may be transfected into cellseither transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified receptors, in which the receptor isexpressed either transiently or in stable cell lines. This inventionfurther provides for a compound identified using a modified receptor ina binding assay such as the binding assays described herein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention also provides an isolated galanin GALR3 receptor protein.In one embodiment, the GALR3 receptor protein has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid K1086. In another embodiment, the GALR3 receptorprotein has the amino acid sequence encoded by plasmid K1086. In anotherembodiment, the protein has the amino acid sequence encoded by theplasmid pEXJ-hGalR3. In an embodiment, the GALR3 receptor protein hasthe same or substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 (Seq. I.D. No. 2). In an embodiment, the GALR3receptor comprises the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 4 (Seq. I.D. No. 4)from amino acid 60 through amino acid 427.

This invention provides a vector comprising the above-described nucleicacid molecule.

Vectors which comprise the isolated nucleic acid molecule describedhereinabove also are provided. Suitable vectors comprise, but are notlimited to, a plasmid or a virus. These vectors may be transformed intoa suitable host cell to form a host cell expression system for theproduction of a polypeptide having the biological activity of a galaninGALR3 receptor. Suitable host cells include, for example, neuronal cellssuch as the glial cell line C6, a Xenopus cell such as an oocyte ormelanophore cell, as well as numerous mammalian cells and non-neuronalcells.

This invention provides the above-described vector adapted forexpression in a bacterial cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the bacterialcell operatively linked to the nucleic acid encoding the GALR3 receptoras to permit expression thereof.

This invention provides the above-described vector adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the yeast celloperatively linked to the nucleic acid encoding the GALR3 receptor as topermit expression thereof.

This invention provides the above-described vector adapted forexpression in an insect cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the insect celloperatively linked to the nucleic acid encoding the GALR3 receptor as topermit expression thereof. In a still further embodiment, the vector isa baculovirus.

This invention provides the above-described vector adapted forexpression in a amphibian cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the amphibiancell operatively linked to the nucleic acid encoding the GALR3 receptoras to permit expression thereof.

In an embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe nucleic acid in the mammalian cell operatively linked to the nucleicacid encoding the mammalian GALR3 receptor as to permit expressionthereof.

In a further embodiment, the vector is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the rat GALR3 receptor as to permitexpression thereof.

In a still further embodiment, the vector is a plasmid.

In another embodiment, the plasmid is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the human GALR3 receptor as to permitexpression thereof.

This invention provides the above-described plasmid adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of nucleic acid in a mammalian cell operativelylinked to the nucleic acid encoding the mammalian GALR3 receptor as topermit expression thereof.

This invention provides a plasmid designated K1086 (ATCC Accession No.97747) which comprises the regulatory elements necessary for expressionof DNA in a mammalian cell operatively linked to DNA encoding the GALR3galanin receptor so as to permit expression thereof.

This plasmid (K1086) was deposited on Oct. 8, 1996, with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. 97747.

This invention provides a plasmid designated pEXJ-hGalR3 (ATCC AccessionNo. 97827) which comprises the regulatory elements necessary forexpression of DNA in a mammalian cell operatively linked to DNA encodingthe human GALR3 galanin receptor so as to permit expression thereof.

This plasmid was deposited Dec. 17, 1996, with the ATCC, 12301 ParklawnDrive, Rockville, Md., 20852, U.S.A. under the provisions of theBudapest Treaty forth International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. 97827.

This invention provides a plasmid designated pEXJ-rGalR3T (ATCCAccession No. 97826) which comprises the regulatory elements necessaryfor expression of DNA in a mammalian cell operatively linked to DNAencoding the rat GALR3 galanin receptor so as to permit expressionthereof.

This plasmid was deposited Dec. 17, 1996, with the ATCC, 12301 ParklawnDrive, Rockville, Md., 20852, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. 97826.

This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the receptor dependingupon the host cell used. In one embodiment, the vector or plasmidcomprises the coding sequence of the GALR3 receptor and the regulatoryelements necessary for expression in the host cell.

This invention provides a eukaryotic cell comprising the above-describedplasmid or vector. This invention provides a mammalian cell comprisingthe above-described plasmid or vector. In an embodiment the cell is aXenopus oocyte or melanophore cell. In an embodiment, the cell is aneuronal cell such as the glial cell line C6. In an embodiment, themammalian cell is non-neuronal in origin. In an embodiment, themammalian cell is a COS-7 cell. In another embodiment the mammalian cellis a Chinese hamster ovary (CHO) cell. In another embodiment, the cellis a mouse Y1 cell.

In still another embodiment, the mammalian cell is a 293 human embryonickidney cell. In still another embodiment, the mammalian cell is aNIH-3T3 cell. In another embodiment, the mammalian cell is an LM(tk-)cell.

In an embodiment, the mammalian cell is the 293 cell designated293-rGALR3-105, which comprises the “trimmed” plasmid pEXJ-rGalR3T. Thiscell line was deposited with the ATCC on Feb. 19, 1997, under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure, andwas accorded ATCC Accession No. CRL-12287.

In an embodiment, the mammalian cell is the LM(tk-) cell designatedL-hGALR3-228, which comprises the plasmid pEXJ-hGalR3. This cell linewas deposited with the ATCC on Jun. 25, 1997, under the provisions ofthe Budapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, and was accordedATCC Accession No. CRL-12373.

This invention also provides an insect cell comprising theabove-described vector. In an embodiment, the insect cell is an Sf9cell. In another embodiment, the insect cell is an Sf21 cell.

This invention provides a membrane preparation isolated from any of theabove-described cells.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR3 receptor contained in plasmid K1086.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR3 receptor contained in plasmidpEXJ-rGalR3T.

This invention still further provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 (Seq. ID NO. 1) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 1 (Seq. ID No. 1).

This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR3 receptor contained in plasmidpEXJ-hGalR3. This invention provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a GALR3 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 3 (Seq. ID No. 3) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 3 (Seq. ID NO. 3).

This invention provides a nucleic acid probe comprising a nucleic acidwhich specifically hybridizes with a nucleic acid encoding a GALR3receptor, wherein the probe comprises a unique sequence of at least 15nucleotides within a fragment of (a) the nucleic acid sequence containedin plasmid K1086 or (b) the antisense nucleic acid sequence capable ofspecifically hybridizing to the nucleic acid sequence contained inplasmid K1086. In one embodiment the GALR3 receptor is encoded by thecoding sequence of the plasmid K1086, or the reverse complement(antisense sequence) of the coding sequence of plasmid K1086. In anembodiment, the nucleic acid encoding a GALR3 receptor comprises anintron.

This invention further provides a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides which is complementaryto a unique fragment of the sequence of a nucleic acid molecule encodinga GALR3 receptor. This invention also provides a nucleic acid probecomprising a nucleic acid molecule of at least 15 nucleotides which iscomplementary to the antisense sequence of a unique fragment of thesequence of a nucleic acid molecule encoding a GALR3 receptor.

In an embodiment, the nucleic acid probe is DNA. In another embodimentthe nucleic acid probe is RNA. As used herein, the phrase “specificallyhybridizing” means the ability of a nucleic acid molecule to recognize anucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs.

This nucleic acid of at least 15 nucleotides capable of specificallyhybridizing with a sequence of a nucleic acid encoding the GALR3 galaninreceptors can be used as a probe. Nucleic acid probe technology is wellknown to those skilled in the art who will readily appreciate that suchprobes may vary greatly in length and may be labeled with a detectablelabel, such as a radioisotope or fluorescent dye, to facilitatedetection of the probe. DNA probe molecules may be produced by insertionof a DNA molecule which encodes the GALR3 receptor into suitablevectors, such as plasmids or bacteriophages, followed by transforminginto suitable bacterial host cells, replication in the transformedbacterial host cells and harvesting of the DNA probes, using methodswell known in the art. Alternatively, probes may be generated chemicallyfrom DNA synthesizers.

RNA probes may be generated by inserting the DNA molecule which encodesthe GALR3 galanin receptor downstream of a bacteriophage promoter suchas T3, T7 or SP6. Large amounts of RNA probe may be produced byincubating the labeled nucleotides with the linearized fragment where itcontains an upstream promoter in the presence of the appropriate RNApolymerase.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a GALR3 galaninreceptor, so as to prevent translation of the mRNA.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to the genomic DNA molecule encodinga GALR3 receptor.

This invention provides an antisense oligonucleotide comprising chemicalanalogues of nucleotides.

This invention provides an antibody directed to a GALR3 receptor. Thisinvention also provides an antibody directed to a rat GALR3 receptor.This invention also provides an antibody directed to a human GALR3receptor. In an embodiment, the rat GALR3 has an amino acid sequencesubstantially the same as an amino acid sequence encoded by plasmidK1086. In an embodiment, the human GALR3 receptor has a sequence, whichsequence comprises substantially the same sequence as the sequence shownin FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.

This invention provides a monoclonal antibody directed to an epitope ofa GALR3 receptor, which epitope is present on the surface of a cellexpressing a GALR3 receptor.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a GALR3receptor by passing through a cell membrane and binding specificallywith mRNA encoding a GALR3 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In an embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

This invention provides the above-described pharmaceutical composition,wherein the pharmaceutically acceptable carrier capable of passingthrough a cell membrane comprises a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type.

This invention provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a GALR3receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a GALR3 receptorand a pharmaceutically acceptable carrier.

This invention provides the above-described pharmaceutical compositionwhich comprises an amount of the antibody effective to block binding ofa ligand to the GALR3 receptor and a pharmaceutically acceptablecarrier.

As used herein, “pharmaceutically acceptable carriers” means any of thestandard pharmaceutically acceptable carriers. Examples include, but arenot limited to, phosphate buffered saline, physiological saline, waterand emulsions, such as oil/water emulsions.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a GALR3 receptor.

This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native GALR3 receptor.

This invention provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a GALR3 receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a GALR3 receptor and which hybridizes tomRNA encoding a GALR3 receptor thereby reducing its translation.

This invention provides the above-described transgenic nonhuman mammal,wherein the DNA encoding a GALR3 receptor additionally comprises aninducible promoter.

This invention provides the transgenic nonhuman mammal, wherein the DNAencoding a GALR3 receptor additionally comprises tissue specificregulatory elements.

In an embodiment, the transgenic nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of GALR3 receptor are produced by creating transgenic animals inwhich the activity of the GALR3 receptor is either increased ordecreased, or the amino acid sequence of the expressed GALR3 receptor isaltered, by a variety of techniques. Examples of these techniquesinclude, but are not limited to: 1) Insertion of normal or mutantversions of DNA encoding a GALR3 receptor, by microinjection,electroporation, retroviral transfection or other means well known tothose skilled in the art, into appropriate fertilized embryos in orderto produce a transgenic animal or 2) Homologous recombination of mutantor normal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these GALR3 receptor sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native GALR3 receptors but does express, for example, aninserted mutant GALR3 receptor, which has replaced the native GALR3receptor in the animal's genome by recombination, resulting inunderexpression of the transporter. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added GALR3 receptors, resulting inoverexpression of the GALR3 receptors.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding a GALR3receptor is purified from a vector by methods well known in the art.Inducible promoters may be fused with the coding region of the DNA toprovide an experimental means to regulate expression of the trans-gene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is inserted into the pronucleus of the egg, and the DNAsolution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a GALR3 receptor which comprises contactingcells containing DNA encoding and expressing on their cell surface theGALR3 receptor, wherein such cells do not normally express the GALR3receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the GALR3receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a GALR3 receptor which comprisescontacting a membrane fraction from a cell extract of cells containingDNA encoding and expressing on their cell surface the GALR3 receptor,wherein such cells do not normally express the GALR3 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the GALR3 receptor.

This invention also provides a process for determining whether achemical compound can specifically bind to a GALR3 receptor whichcomprises contacting cells transfected with and expressing DNA encodingthe GALR3 receptor with the compound under conditions permitting bindingof compounds to such receptor, and detecting the presence of any suchcompound specifically bound to the GALR3 receptor, so as to therebydetermine whether the ligand specifically binds to the GALR3 receptor.

This invention provides a process for determining whether a chemicalcompound can specifically bind to a GALR3 receptor which comprisespreparing a cell extract from cells transfected with and expressing DNAencoding the GALR3 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the compound underconditions permitting binding of compounds to such receptor, anddetecting the presence of the compound specifically bound to the GALR3receptor, so as to thereby determine whether the compound specificallybinds to the GALR3 receptor.

In one embodiment, the GALR3 receptor is a mammalian GALR3 receptor. Inanother embodiment, the GALR3 receptor is a rat GALR3 receptor. In stillanother embodiment, the GALR3 receptor has the same or substantially thesame amino acid sequence as that encoded by plasmid K1086. In stillanother embodiment, the GALR3 receptor has the amino acid sequenceencoded by plasmid K1086. In another embodiment, the GALR3 receptor hassubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 2 (Seq. ID NO. 2). In another embodiment, the GALR3receptor has the amino acid sequence shown in FIG. 2 (Seq. ID NO. 2). Instill another embodiment, the cells are transfected with the plasmidpEXJ-RGALR3T (ATCC Accession No. 97826), encoding the rat GALR3receptor. Plasmid pEXJ-RGalR3T comprises the entire coding region of ratGALR3, but in which the 5′ initiating ATG is joined directly to thevector, and which comprises only 100 nucleotides from the 3′untranslated region after the stop codon (i.e., up to and includingnucleotide 1275 in FIG. 1 (Seq. ID NO. 1)). Transfection of cells withthe “trimmed” plasmid results in a higher level of expression of the ratGALR3 receptor than the level of expression when plasmid K1086 is used.The use of the “trimmed” plasmid provides for greater convenience andaccuracy in binding assays. In another embodiment the GALR3 receptor isa human GALR3 receptor. In still another embodiment, the GALR3 receptorhas the same or substantially the same amino acid sequence as thatencoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In anembodiment, the human GALR3 receptor has a sequence, which sequencecomprises substantially the same amino acid sequence as the sequenceshown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid427. In another embodiment, the GALR3 receptor has a sequence, whichsequence comprises the sequence shown in FIG. 4 (Seq. ID NO. 4) fromamino acid 60 through amino acid 427.

In an embodiment, the above process further comprises determiningwhether the compound selectively binds to the GALR3 receptor relative toanother galanin receptor. In another embodiment, the determinationwhether the compound selectively binds to the GALR3 receptor comprises:(a) determining the binding affinity of the compound for the GALR3receptor and for such other galanin receptor; and (b) comparing thebinding affinities so determined, the presence of a higher bindingaffinity for the GALR3 receptor than for such other galanin receptorindicating that the compound selectively binds to the GALR3 receptor. Inone embodiment, the other galanin receptor is a GALR1 receptor. Inanother embodiment, the other galanin receptor is a GALR2 receptor.

This invention provides a process for determining whether a chemicalcompound is a GALR3 receptor agonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR3 receptor with thecompound under conditions permitting the activation of the GALR3receptor, and detecting an increase in GALR3 receptor activity, so as tothereby determine whether the compound is a GALR3 receptor agonist.

This invention provides a process for determining whether a chemicalcompound is a GALR3 receptor agonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR3 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting the activation of the GALR3 receptor, and detecting anincrease in GALR3 receptor activity, so as to thereby determine whetherthe compound is a GALR3 receptor agonist.

In one embodiment, the GALR3 receptor is a rat GALR3 receptor. Inanother embodiment, the GALR3 receptor has the same or substantially thesame amino acid sequence as that encoded by the plasmid K1086. In yetanother embodiment, the GALR3 receptor has the amino acid sequenceencoded by the plasmid K1086. In another embodiment, the GALR3 receptorhas substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 (Seq. ID No. 2). In another embodiment, theGALR3 receptor has the amino acid sequence shown in FIG. 2 (Seq. ID No.2). In another embodiment, the GALR3 receptor is a human GALR3 receptor.In still another embodiment, the GALR3 receptor has the same orsubstantially the same amino acid sequence as that encoded by plasmidpEXJ-hGalR3 (ATCC Accession No. 97827). In another embodiment, the humanGALR3 receptor has a sequence, which sequence comprises substantiallythe same amino acid sequence as the sequence shown in FIG. 4 (Seq. I.D.No. 4) from amino acid 60 through amino acid 427. In another embodiment,the GALR3 receptor has a sequence, which sequence comprises the sequenceshown in FIG. 4 (Seq. ID NO. 4) from amino acid 60 through amino acid427. In another embodiment of this invention the cells are transfectedwith plasmid pEXJ-RGalR3T (ATCC Accession No. 97826).

This invention provides a process for determining whether a chemicalcompound is a GALR3 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR3 receptor with thecompound in the presence of a known GALR3 receptor agonist, such asgalanin, under conditions permitting the activation of the GALR3receptor, and detecting a decrease in GALR3 receptor activity, so as tothereby determine whether the compound is a GALR3 receptor antagonist.

This invention provides a process for determining whether a chemicalcompound is a GALR3 receptor antagonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR3 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand in the presence of aknown GALR3 receptor agonist, such as galanin, under conditionspermitting the activation of the GALR3 receptor, and detecting adecrease in GALR3 receptor activity, so as to thereby determine whetherthe compound is a GALR3 receptor antagonist.

In an embodiment, the GALR3 receptor is a mammalian GALR3 receptor. Inone embodiment of the invention, the GALR3 receptor is a rat GALR3receptor. In another embodiment, the GALR3 receptor has the same orsubstantially the same amino acid sequence as that encoded by theplasmid K1086. In still another embodiment, the GALR3 receptor has theamino acid sequence encoded by the plasmid K1086. In another embodiment,the GALR3 receptor has substantially the same amino acid sequence as theamino acid sequence shown in FIG. 2 (Seq. ID No. 2). In anotherembodiment, the GALR3 receptor has the amino acid sequence shown in FIG.2 (Seq. ID No. 2). In another embodiment, the GALR3 receptor is a humanGALR3 receptor. In still another embodiment, the GALR3 receptor has thesame or substantially the same amino acid sequence as that encoded byplasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In another embodiment,the human GALR3 receptor has a sequence, which sequence comprisessubstantially the same amino acid sequence as the sequence shown in FIG.4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427. Inanother embodiment, the GALR3 receptor has a sequence, which sequencecomprises the sequence shown in FIG. 4 (Seq. ID NO. 4) from amino acid60 through amino acid 427.

In an embodiment of the above-described methods, the cell is anon-mammalian cell such as an insect cell or a Xenopus cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, thecell is non-neuronal in origin. In still further embodiments, thenon-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell,NIH-3T3 cell, a CHO cell, or LM(tk-) cell. In another embodiment, thecell is a mouse Y1 cell.

This invention provides a compound determined by the above-describedmethods. In one embodiment of the above-described methods, the compoundis not previously known to bind to a GALR3 receptor.

This invention provides a GALR3 agonist determined by theabove-described methods. This invention also provides a GALR3 antagonistdetermined by the above-described methods.

In an embodiment of any of the above processes, the cells aretransfected with and expressing GIRK1 and GIRK4.

In an embodiment of any of the above processes, the GALR3 receptor is amammalian GALR3 receptor.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor agonist determined by the above-describedprocesses effective to increase activity of a GALR3 receptor and apharmaceutically acceptable carrier. In an embodiment, the GALR3receptor agonist is not previously known.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor antagonist determined by the above-describedprocesses effective to reduce activity of a GALR3 receptor and apharmaceutically acceptable carrier. In an embodiment, the GALR3receptor antagonist is not previously known.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor agonist effective to increase activity of aGALR3 receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor antagonist effective to reduce activity of aGALR3 receptor and a pharmaceutically acceptable carrier.

In further embodiments of the above-described processes, the agonist orantagonist is not previously known to bind to a GALR3 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a GALR3receptor which comprises separately contacting cells expressing on theircell surface the GALR3 receptor, wherein such cells do not normallyexpress the GALR3 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe GALR3 receptor, a decrease in the binding of the second chemicalcompound to the GALR3 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the GALR3 receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to a humanGALR3 receptor which comprises separately contacting a membrane fractionfrom a cell extract of cells expressing on their cell surface the GALR3receptor, wherein such cells do not normally express the GALR3 receptor,with both the chemical compound and a second chemical compound known tobind to the receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the GALR3 receptor, adecrease in the binding of the second chemical compound to the GALR3receptor in the presence of the chemical compound indicating that thechemical compound binds to the GALR3 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a GALR3 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the GALR3 receptor, wherein suchcells do not normally express the GALR3 receptor, with the chemicalcompound under conditions suitable for activation of the GALR3 receptor,and measuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the GALR3 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a GALR3 receptor,which comprises contacting a membrane fraction from a cell extract ofcells producing a second messenger response and expressing on their cellsurface the GALR3 receptor, wherein such cells do not normally expressthe GALR3 receptor, with the chemical compound under conditions suitablefor activation of the GALR3 receptor, and measuring the second messengerresponse in the presence and in the absence of the chemical compound, achange in the second messenger response in the presence of the chemicalcompound indicating that the compound activates the GALR3 receptor.

In an embodiment of the above processes, the second messenger responsecomprises potassium channel activation and the change in secondmessenger is an increase in the level of inward potassium current.

In one embodiment of the above processes, the second messenger responsecomprises adenylate cyclase activity and the change in second messengerresponse is a decrease in adenylate cyclase activity. In an embodiment,adenylate cyclase activity is determined by measurement of cyclic AMPlevels.

In another embodiment of the above processes, the second messengerresponse comprises arachidonic acid release and the change in secondmessenger response is an increase in arachidonic acid levels.

In another embodiment of the above processes, the second messengerresponse comprises intracellular calcium levels and the change in secondmessenger response is an increase in intracellular calcium levels.

In a still further embodiment of the above processes, the secondmessenger response comprises inositol phospholipid hydrolysis and thechange in second messenger response is an increase in inositolphospholipid hydrolysis.

This invention further provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR3 receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface the GALR3receptor, wherein such cells do not normally express the GALR3 receptor,with both the chemical compound and a second chemical compound known toactivate the GALR3 receptor, and with only the second compound, underconditions suitable for activation of the GALR3 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR3 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR3 receptor, which comprises separately contacting a membranefraction from a cell extract of cells producing a second messengerresponse and expressing on their cell surface the GALR3 receptor,wherein such cells do not normally express the GALR3 receptor, with boththe chemical compound and a second chemical compound known to activatethe GALR3 receptor, and with only the second chemical compound, underconditions suitable for activation of the GALR3 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR3 receptor.

In an embodiment of the above processes, the second messenger responsecomprises potassium channel activation and the change in secondmessenger response is a smaller increase in the level of inwardpotassium current in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound.

In one embodiment of the above processes, the second messenger responsecomprises adenylate cyclase activity and the change in second messengerresponse is a smaller decrease in the level of adenylate cyclaseactivity in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In an embodiment, adenylate cyclase activity is determined bymeasurement of cyclic AMP levels.

In another embodiment of the above processes the second messengerresponse comprises arachidonic acid release, and the change in secondmessenger response is a smaller increase in arachidonic acid levels inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compound.

In another embodiment of the above processes the second messengerresponse comprises intracellular calcium levels, and the change insecond messenger response is a smaller increase in intracellular calciumlevels in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In yet another embodiment of the above processes, the second messengerresponse comprises inositol phospholipid hydrolysis, and the change insecond messenger response is a smaller increase in inositol phospholipidhydrolysis in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In an embodiment of any of the above processes, the GALR3 receptor is amammalian GALR3 receptor. In another embodiment of the above processes,the GALR3 receptor is a rat GALR3 receptor or a human GALR3 receptor. Instill another embodiment of the above processes, the GALR3 receptor hasthe same or substantially the same amino acid sequence as encoded by theplasmid K1086 (ATCC Accession No. 97747). In another embodiment, theGALR3 receptor has substantially the same amino acid sequence as theamino acid sequence shown in FIG. 2 (Seq. ID No. 2). In anotherembodiment, the GALR3 receptor has the amino acid sequence shown in FIG.2 (Seq. ID No. 2). In still another embodiment, the GALR3 receptor hasthe same or substantially the same amino acid sequence as that encodedby plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In anotherembodiment, the human GALR3 receptor has a sequence, which sequencecomprises substantially the same amino acid sequence as the sequenceshown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid427. In another embodiment, the GALR3 receptor has a sequence, whichsequence comprises the sequence shown in FIG. 4 (Seq. ID NO. 4) fromamino acid 60 through amino acid 427. In another embodiment of thisinvention the cells are transfected with plasmid pEXJ-RGalR3T (ATCCAccession No. 97826).

In one embodiment of the above-described processes, the cell is anon-mammalian cell such as an insect cell or a Xenopus cell. In anotherembodiment of any of the above processes, the cell is a mammalian cell.In still further embodiments, the cell is nonneuronal in origin. Inanother embodiment of the above processes, the nonneuronal cell is aCOS-7 cell, 293 human embryonic kidney cell, CHO cell, mouse Y1 cell,NIH-3T3 cell or LM(tk-) cell.

This invention further provides a compound determined by any of theabove processes. In another embodiment, the compound is not previouslyknown to bind to a GALR3 receptor.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor agonist determined by any of the aboveprocesses effective to increase activity of a GALR3 receptor and apharmaceutically acceptable carrier. In an embodiment, the GALR3receptor agonist is not previously known.

This invention provides a pharmaceutical composition which comprises anamount of a GALR3 receptor antagonist determined by any of the aboveprocesses effective to reduce activity of a GALR3 receptor and apharmaceutically acceptable carrier. In an embodiment, the GALR3receptor antagonist is not previously known.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR3 receptor to identify a compoundwhich specifically binds to the GALR3 receptor, which comprises (a)contacting cells transfected with and expressing DNA encoding the GALR3receptor with a compound known to bind specifically to the GALR3receptor; (b) contacting the preparation of step (a) with the pluralityof compounds not known to bind specifically to the GALR3 receptor, underconditions permitting binding of compounds known to bind the GALR3receptor; (c) determining whether the binding of the compound known tobind to the GALR3 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to theGALR3 receptor of each compound included in the plurality of compounds,so as to thereby identify the compound which specifically binds to theGALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR3 receptor to identify a compoundwhich specifically binds to the GALR3 receptor, which comprises (a)preparing a cell extract from cells transfected with and expressing DNAencoding the GALR3 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with a compound known to bindspecifically to the GALR3 receptor; (b) contacting the preparation ofstep (a) with the plurality of compounds not known to bind specificallyto the GALR3 receptor, under conditions permitting binding of compoundsknown to bind the GALR3 receptor; (c) determining whether the binding ofthe compound known to bind to the GALR3 receptor is reduced in thepresence of the compounds, relative to the binding of the compound inthe absence of the plurality of compounds; and if so (d) separatelydetermining the binding to the GALR3 receptor of each compound includedin the plurality of compounds, so as to thereby identify the compoundwhich specifically binds to the GALR3 receptor.

In an embodiment of any of the above processes, the GALR3 receptor is amammalian GALR3 receptor. In an embodiment of the above-describedmethods, the GALR3 receptor is a rat GALR3 receptor. In anotherembodiment, the GALR3 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence encoded by plasmid K1086.In another embodiment, the GALR3 receptor has substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 2 (Seq. IDNO. 2). In another embodiment, the GALR3 receptor has the amino acidsequence shown in FIG. 2 (Seq. ID No. 2). In another embodiment, theGALR3 receptor is a human GALR3 receptor. In still another embodiment,the GALR3 receptor has the same or substantially the same amino acidsequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No.97827). In another embodiment, the human GALR3 receptor has a sequence,which sequence comprises substantially the same amino acid sequence asthe sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60through amino acid 427. In another embodiment, the GALR3 receptor has asequence, which sequence comprises the sequence shown in FIG. 4 (Seq. IDNO. 4) from amino acid 60 through amino acid 427.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a GALR3 receptor to identify a compoundwhich activates the GALR3 receptor which comprises (a) contacting cellstransfected with and expressing the GALR3 receptor with the plurality ofcompounds not known to activate the GALR3 receptor, under conditionspermitting activation of the GALR3 receptor; (b) determining whether theactivity of the GALR3 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the GALR3 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the GALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a GALR3 receptor to identify a compoundwhich activates the GALR3 receptor which comprises (a) preparing a cellextract from cells transfected with and expressing DNA encoding theGALR3 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the plurality of compounds notknown to activate the GALR3 receptor, under conditions permittingactivation of the GALR3 receptor; (b) determining whether the activityof the GALR3 receptor is increased in the presence of the compounds; andif so (c) separately determining whether the activation of the GALR3receptor is increased by each compound included in the plurality ofcompounds, so as to thereby identify the compound which activates theGALR3 receptor.

In an embodiment of the above processes, the cells are transfected withand expressing GIRK1 and GIRK4. In another embodiment, the GALR3receptor is a mammalian GALR3 receptor.

In an embodiment of any of the above-described methods, the GALR3receptor is a rat GALR3 receptor. In still another embodiment, the GALR3receptor has the same or substantially the same amino acid sequence asthe amino acid sequence encoded by plasmid K1086. In another embodiment,the GALR3 receptor has substantially the same amino acid sequence as theamino acid sequence shown in FIG. 2 (Seq. ID No. 2). In anotherembodiment, the GALR3 receptor has the amino acid sequence shown in FIG.2 (Seq. ID No. 2). In another embodiment, the GALR3 receptor is a humanGALR3 receptor. In still another embodiment, the GALR3 receptor has thesame or substantially the same amino acid sequence as that encoded byplasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In another embodiment,the human GALR3 receptor has a sequence, which sequence comprisessubstantially the same amino acid sequence as the sequence shown in FIG.4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427. Inanother embodiment, the GALR3 receptor has a sequence, which sequencecomprises the sequence shown in FIG. 4 (Seq. ID NO. 4) from amino acid60 through amino acid 427.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a GALR3 receptor toidentify a compound which inhibits the activation of the GALR3 receptor,which comprises (a) contacting cells transfected with and expressing theGALR3 receptor with the plurality of compounds in the presence of aknown GALR3 receptor agonist, under conditions permitting activation ofthe GALR3 receptor; (b) determining whether the activation of the GALR3receptor is reduced in the presence of the plurality of compounds,relative to the activation of the GALR3 receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the GALR3 receptor for each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which inhibits the activation of the GALR3 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a GALR3 receptor toidentify a compound which inhibits the activation of the GALR3 receptor,which comprises (a) preparing a cell extract from cells transfected withand expressing DNA encoding the GALR3 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe plurality of compounds in the presence of a known GALR3 receptoragonist, under conditions permitting activation of the GALR3 receptor;(b) determining whether the activation of the GALR3 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the GALR3 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the GALR3 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the GALR3 receptor.

In an embodiment of the above processes, the cells are transfected withand expressing GIRK1 and GIRK4. In another embodiment, the GALR3receptor is a mammalian GALR3 receptor.

In an embodiment of any of the above-described methods, the GALR3receptor is a rat GALR3 receptor. In another embodiment, the GALR3receptor has the same or substantially the same amino acid sequence asthe amino acid sequence encoded by plasmid K1086. In another embodiment,the GALR3 receptor has substantially the same amino acid sequence as theamino acid sequence shown in FIG. 2 (Seq. ID No. 2). In anotherembodiment, the GALR3 receptor has the amino acid sequence shown in FIG.2 (Seq. ID No. 2). In another embodiment, the GALR3 receptor is a humanGALR3 receptor. In still another embodiment, the GALR3 receptor has thesame or substantially the same amino acid sequence as that encoded byplasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In another embodiment,the human GALR3 receptor has a sequence, which sequence comprisessubstantially the same amino acid sequence as the sequence shown in FIG.4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427. Inanother embodiment, the GALR3 receptor has a sequence, which sequencecomprises the sequence shown in FIG. 4 (Seq. ID NO. 4) from amino acid60 through amino acid 427.

In an embodiment of the above processes, the cells are transfected withand expressing GIRK1 and GIRK4. In an embodiment of the above processes,receptor activation is determined by measurement of potassium channelactivation. In an embodiment, receptor activation is determined bymeasurement of an increase in inward potassium current. In anotherembodiment, inhibition of receptor activation is determined by a smallerincrease in inward potassium current in the presence of the compound anda galanin receptor agonist than in the presence of only the galaninreceptor agonist. In an embodiment, the galanin receptor agonist isgalanin.

This invention provides a pharmaceutical composition comprising acompound identified by any of the above-described methods effective toincrease GALR3 receptor activity and a pharmaceutically acceptablecarrier.

This invention provides a pharmaceutical composition comprising acompound identified by any of the above-described methods effective todecrease GALR3 receptor activity and a pharmaceutically acceptablecarrier.

This invention provides any of the above processes, which furthercomprises a process for determining whether the compound selectivelyactivates the GALR3 receptor relative to another galanin receptor.

This invention provides a process for determining whether a compoundselectively activates the GALR3 receptor relative to another galaninreceptor which comprises: (a) determining the potency of the compoundfor the GALR3 receptor and for such other galanin receptor; and (b)comparing the potencies so determined, the presence of a higher potencyfor the GALR3 receptor than for such other galanin receptor indicatingthat the compound selectively activates the GALR3 receptor. In anembodiment of the above process such other galanin receptor is a GALR1receptor. In another embodiment, such other galanin receptor is a GALR2receptor.

This invention further provides any of the above processes, whichfurther comprises a process for determining whether the compoundselectively inhibits the activation of the GALR3 receptor relative toanother galanin receptor.

This invention provides a process for determining whether a compoundselectively inhibits the activation of the GALR3 receptor relative toanother galanin receptor, which comprises: (a) determining the decreasein the potency of a known galanin receptor agonist for the GALR3receptor in the presence of the compound, relative to the potency of theagonist in the absence of the compound; (b) determining the decrease inthe potency of the agonist for such other galanin receptor in thepresence of the compound, relative to the potency of the agonist in theabsence of the compound; and (c) comparing the decrease in potencies sodetermined, the presence of a greater decrease in potency for the GALR3receptor than for such other galanin receptor indicating that thecompound selectively inhibits the activation of the GALR3 receptor. Inan embodiment of the above processes, such other galanin receptor is aGALR1 receptor. In another embodiment, such other galanin receptor is aGALR2 receptor.

In an embodiment of any of the above-described methods, the activationof the GALR3 receptor is determined by a second messenger assay. In anembodiment, the second messenger assay measures adenylate cyclaseactivity. In other embodiments, the second messenger is cyclic AMP,intracellular calcium, or arachidonic acid or a phosphoinositol lipidmetabolite. Receptor activation may also be measured by assaying thebinding of GTPTS (gamma thiol GTP) to membranes, which precedes and istherefore independent of second messenger coupling.

This invention further provides a method of measuring GALR3 receptoractivation in an oocyte expression system such as a Xenopus oocyte ormelanophore. In an embodiment, receptor activation is determined bymeasurement of ion channel activity, e.g., using the voltage clamptechnique (Stühmer, 1992). In an embodiment, receptor activation isdetermined by the measurement of inward potassium current. In theexperiments described hereinbelow, receptor activation was determined bymeasurement of inward potassium current in the presence of elevatedexternal potassium levels. However, this invention also provides amethod of determining GALR3 receptor activation by measurement ofoutward potassium current in the presence of low (i.e., physiologic)external potassium levels, using similar methods, which are well-knownin the art.

Expression of genes in Xenopus oocytes is well known in the art (A.Coleman, Transcription and Translation: A Practical Approach (B. D.Hanes, S. J. Higgins, eds., pp 271-302, IRL Press, Oxford, 1984; Y. Masuet al., Nature 329:21583-21586, 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989) including using T7 polymerase with the mCAPRNA capping kit (Stratagene). The use of DNA vectors that include 5′ and3′ untranslated (UT) regions of Xenopus β-globin gene flanking thecoding region of the gene of interest has been found to increase thelevel of expression in Xenopus oocytes (Linman, et al., 1992).

In an embodiment of any of the above-described processes or methods, thecell is a non-mammalian cell such as an insect cell or Xenopus cell. Ina further embodiment of the invention, the cell is a mammalian cell. Inanother embodiment of the invention, the mammalian cell is non-neuronalin origin. In still further embodiments of the invention, thenon-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, aLM(tk-) cell, a mouse Y1 cell, a CHO cell, or an NIH-3T3 cell.

This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods and apharmaceutically acceptable carrier.

In an embodiment of the above-described methods, the cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, 293 human embryonic kidney cell, CHO cell, NIH-3T3 cellor LM(tk-) cell.

In one embodiment of the above-described methods, the compound is notpreviously known to bind to a GALR3 receptor.

This invention provides a GALR3 receptor agonist detected by theabove-described methods. This invention provides a GALR3 receptorantagonist detected by the above-described methods. In an embodiment thecell is a non-mammalian cell, for example, a Xenopus oocyte ormelanophore. In another embodiment the cell is a neuronal cell, forexample, a glial cell line such as C6. In an embodiment, the cell isnon-neuronal in origin. In a further embodiment, the cell is a Cos-7 ora CHO cell, a 293 human embryonic kidney cell, an LM(tk-) cell or anNIH-3T3 cell.

This invention provides a pharmaceutical composition comprising a drugcandidate identified by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method for determining whether a chemicalcompound is a GALR3 antagonist which comprises: (a) administering to ananimal a GALR3 agonist and measuring the amount of food intake in theanimal; (b) administering to a second animal both the GALR3 agonist andthe chemical compound, and measuring the amount of food intake in thesecond animal; and (c) determining whether the amount of food intake isreduced in the presence of the chemical compound relative to the amountof food intake in the absence of the compound, so as to therebydetermine whether the compound is a GALR3 antagonist. This inventionfurther provides a method of screening a plurality of chemical compoundsto identify a chemical compound which is a GALR3 antagonist whichcomprises: (a) administering to an animal a GALR3 agonist and measuringthe amount of food intake in the animal; (b) administering to a secondanimal the GALR3 agonist and at least one chemical compound of theplurality of compounds, and measuring the amount of food intake in theanimal; (c) determining whether the amount of food intake is reduced inthe presence of at least one chemical compound of the plurality ofchemical compounds relative to the amount of food intake in the absenceof at least one of the compounds, and if so; (d) separately determiningwhether each chemical compound is a GALR3 antagonist according to themethod described above, so as to thereby determine it the chemicalcompound is a GALR3 antagonist. In another embodiment the animal is anon-human mammal. In a further embodiment, the animal is a rodent.

This invention provides a method of detecting expression of a GALR3receptor by detecting the presence of mRNA coding for the GALR3 receptorwhich comprises obtaining total mRNA from a cell or tissue sample andcontacting the mRNA so obtained with the above-described nucleic acidprobe under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of theGALR3 receptor by the cell or in the tissue.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by administering to thesubject an amount of a GALR3 selective compound, effective to treat theabnormality. Abnormalities which may be treated include cognitivedisorder, pain, sensory disorder (olfactory, visual), motor coordinationabnormality, motion sickness, neuroendocrine disorders, sleep disorders,migraine, Parkinson's disease, hypertension, heart failure,convulsion/epilepsy, traumatic brain injury, diabetes, glaucoma,electrolyte imbalances, respiratory disorders (asthma, emphysema),depression, reproductive disorders, gastric and intestinal ulcers,gastroesophageal reflux disorder, gastric hypersecretion,gastrointestinal motility disorders (diarrhea), inflammation, immunedisorders, and anxiety. In one embodiment the compound is an agonist. Inanother embodiment the compound is an antagonist.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of aGALR3 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective todecrease the activity of the GALR3 receptor in the subject, therebytreating the abnormality in the subject. In an embodiment, theabnormality is obesity. In another embodiment, the abnormality isbulimia.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a GALR3receptor which comprises administering to a subject an effective amountof the above-described pharmaceutical composition effective to activatethe GALR3 receptor in the subject. In an embodiment, the abnormalcondition is anorexia.

In another embodiment, the compound binds selectively to a GALR3receptor. In yet another embodiment, the compound binds to the GALR3receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to a GALR1 receptor. In a still furtherembodiment, the compound binds to the GALR3 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to a GALR2 receptor.

This invention provides a method of detecting the presence of a GALR3receptor on the surface of a cell which comprises contacting the cellwith the above-described antibody under conditions permitting binding ofthe antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of a GALR3receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR3 receptors which comprisesproducing a transgenic nonhuman mammal whose levels of GALR3 receptoractivity are varied by use of an inducible promoter which regulatesGALR3 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR3 receptors which comprisesproducing a panel of transgenic nonhuman mammals each expressing adifferent amount of GALR3 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a GALR3 receptor comprising administering acompound to the above-described transgenic nonhuman mammal anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofover activity of a GALR3 receptor, the alleviation of the abnormalityidentifying the compound as an antagonist.

This invention provides an antagonist identified by the above-describedmethods. This invention provides a pharmaceutical composition comprisingan antagonist identified by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of aGALR3 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition, therebytreating the abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a GALR3 receptor comprisingadministering a compound to a transgenic nonhuman mammal and determiningwhether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.

This invention provides an agonist identified by the above-describedmethods.

This invention provides a pharmaceutical composition comprising anagonist identified by the above-described methods and a pharmaceuticallyacceptable carrier.

This invention provides a method for treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a GALR3 receptor which comprises administering to a subject aneffective amount of the above-described pharmaceutical composition,thereby treating the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific human GALR3 receptorallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human GALR3 receptor and labelled with a detectable marker;(e) detecting labelled bands which have hybridized to DNA encoding ahuman GALR3 receptor labelled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps a-e; and (g)comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

In an embodiment, a disorder associated with the activity of a specifichuman GALR3 receptor allele is diagnosed.

In another embodiment, the above-described method may be used toidentify a population of patients having a specific GALR3 receptorallele, in which population the disorder may be alleviated byadministering to the subjects a GALR3-selective compound.

This invention provides a method of preparing the purified GALR3receptor which comprises: (a) inducing cells to express GALR3 receptor;(b) recovering the receptor from the induced cells; and (c) purifyingthe receptor so recovered.

This invention provides a method of preparing a purified GALR3 receptorwhich comprises: (a) inserting nucleic acid encoding the GALR3 receptorin a suitable vector; (b) introducing the resulting vector in a suitablehost cell; (c) placing the resulting cell in suitable conditionpermitting the production of the isolated GALR3 receptor; (d) recoveringthe receptor produced by the resulting cell; and (e) purifying thereceptor so recovered.

This invention provides a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In one embodiment, thecompound is a GALR3 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In another embodimentthe compound is administered in combination with food.

In yet another embodiment the compound is a GALR3 receptor agonist andthe amount is effective to increase the consumption of food by thesubject. In a still further embodiment, the compound is administered incombination with food. In other embodiments the subject is a vertebrate,a mammal, a human or a canine.

In one embodiment, the compound binds selectively to a GALR3 receptor.In another embodiment, the compound binds to the GALR3 receptor with anaffinity greater than ten-fold higher than the affinity with which thecompound binds to a GALR1 receptor. In another embodiment, the compoundbinds to the GALR3 receptor with an affinity greater than ten-foldhigher than the affinity with which the compound binds to a GALR2receptor. In yet another embodiment, the compound binds to the GALR3receptor with an affinity greater than one hundred-fold higher than theaffinity with which the compound binds to a GALR1 receptor. In anotherembodiment, the compound binds to the GALR3 receptor with an affinitygreater than one hundred-fold higher than the affinity with which thecompound binds to a GALR2 receptor.

This invention provides a method of treating Alzheimer's disease in asubject which comprises administering to the subject an amount of acompound which is a galanin receptor antagonist effective to treat thesubject's Alzheimer's disease. In one embodiment, the galanin receptorantagonist is a GALR3 receptor antagonist and the amount of the compoundis effective to treat the subject's Alzheimer's disease.

This invention provides a method of producing analgesia in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a galanin receptor agonist effective to produce analgesia inthe subject. In another embodiment, the galanin receptor agonist is aGAUR3 receptor agonist and the amount of the compound is effective toproduce analgesia in the subject.

This invention provides a method of decreasing nociception in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a GALR3 receptor agonist effective to decrease nociception inthe subject.

This invention provides a method of treating pain in a subject whichcomprises administering to the subject an amount of a compound which isa GALR3 receptor agonist effective to treat pain in the subject.

This invention provides a method of treating diabetes in a subject whichcomprises administering to the subject an amount of a compound which isa GALR3 receptor antagonist effective to treat diabetes in the subject.

This invention provides a method of decreasing feeding behavior of asubject which comprises administering a compound which is a GALR3receptor antagonist and a compound which is a Y5 receptor antagonist,the amount of such antagonists being effective to decrease the feedingbehavior of the subject. In an embodiment, the GALR3 antagonist and theY5 antagonist are administered in combination. In another embodiment,the GALR3 antagonist and the Y5 antagonist are administered once. Inanother embodiment, the GALR3 antagonist and the Y5 antagonist areadministered separately. In still another embodiment, the GALR3antagonist and the Y5 antagonist are administered once. In anotherembodiment, the galanin receptor antagonist is administered for about 1week to 2 weeks. In another embodiment, the Y5 receptor antagonist isadministered for about 1 week to 2 weeks.

In yet another embodiment, the GALR3 antagonist and the Y5 antagonistare administered alternately. In another embodiment, the GALR3antagonist and the Y5 antagonist are administered repeatedly. In a stillfurther embodiment, the galanin receptor antagonist is administered forabout 1 week to 2 weeks. In another embodiment, the Y5 receptorantagonist is administered for about 1 week to 2 weeks. This inventionalso provides a method as described above, wherein the compound isadministered in a pharmaceutical composition comprising a sustainedrelease formulation.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods Cloninc and Sequencing aNovel Rat Galanin Receptor Fragment

A rat hypothalamus cDNA library in lambda ZAP II (≈2.5×10⁶ totalrecombinants; Stratagene, LaJolla, Calif.) was screened usingoverlapping transmembrane (TM) oligonucleotide probes (TM 1, 2, 3, 4, 5,6 and 7) derived from the rat GALR2 receptor cDNA. Overlapping oligomerswere labeled with [³²P] DATP and [³²P] dCTP by synthesis with the largefragment of DNA polymerase, and comprised the following sequences:

TM1:

(+)strand: 5′TTGTACCCCTATTTTTCGCGCTCATCTTCCTCGTGGGCACCGTGG-3′ (SEQ IDNO: 6);

(−)strand: 5′-AGCACCGCCAGCACCAGCGCGTTGCCCACGGTGCCCACGAGGAAG-3′ (SEQ IDNO: 7);

TM2:

(+)strand: 5′-TCAGCACCACCAACCTGTTCATCCTCAACCTGGGCGTGGCCGACCTGTGT-3′ (SEQID NO: 8);

(−)strand: 5′-GGCCTGGAAAGGCACGCAGCACAGGATGAAACACAGGTCGGCCACGCCCA-3′ (SEQID NO: 9);

TM3:

(+)strand: 5′-CTGCAAGGCTGTTCATTTCCTCATCTTTCTCACTATGCACGCCAG-3′ (SEQ IDNO: 10);

(−)strand: 5′-GGAGACGGCGGCCAGCGTGAAGCTGCTGGCGTGCATAGTGAGAAA-3′ (SEQ IDNO: 11);

TM4:

(+)strand 5′-AACGCGCTGGCCGCCATCGGGCTCATCTGGGGGCTAGCACTGCTC-3′ (SEQ IDNO: 12);

(−)strand 5′-AGTAGCTCAGGTAGGGCCCGGAGAAGAGCAGTGCTAGCCCCCAGA-3′ (SEQ IDNO: 13);

TM5:

(+)strand: 5′-AGCCATGGACCTCTGCACCTTCGTCTTTAGCTACCTGCTGCCAGT-3′ (SEQ IDNO: 14);

(−)strand: 5′-CGCATAGGTCAGACTGAGGACTAGCACTGGCAGCAGGTAGCTAAA-3′ (SEQ IDNO: 15);

TM6:

(+)strand: 5′-GATCATCATCGTGGCGGTGCTTTTCTGCCTCTGTTGGATGCCCCA-3′ (SEQ IDNO: 16);

(−)strand: 5′-CCACACGCAGAGGATAAGCGCGTGGTGGGGCATCCAACAGAGGCA-3′ (SEQ IDNO: 17);

TM7:

(+)strand: 5′-GTTGCGCATCCTTTCACACCTAGTTTCCTATGCCAACTCCTGTGT-3′ (SEQ IDNO: 18);

(−)strand: 5′-AGACCAGAGCGTAAACGATGGGGTTGACACAGGAGTTGGCATAGGA-3′ (SEQ IDNO: 19).

Hybridization of phage lifts was performed at reduced stringencyconditions: 40° C. in a solution containing 37.5% formamide, 5×SSC(1×SSC is 0.15M sodium chloride, 0.015M sodium citrate), 1×Denhardt'ssolution (0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serumalbumin), and 25 μg/μL sonicated salmon sperm DNA. The filters werewashed at 45° C. in 0.1×SSC containing 0.1% sodium dodecyl sulfate andexposed at −70° C. to Kodak BioMax film in the presence of anintensifying screen. Lambda phage clones hybridizing with the probeswere plaque purified and pBluescript recombinant DNAs wereexcision-rescued from λ Zap II using helper phage Re704, as described bythe manufacturer's protocol (Rapid Excision Kit, Stratagene, LaJolla,Calif.). Insert size was confirmed by restriction enzyme digestanalysis. The cDNA insert was sequenced on both strands by cyclesequencing with AmpliTaq DNA Polymerase, FS (Perkin Elmer) and productsrun on an automated fluorescent sequencer, the ABI Prism 377 Sequencer(ABI). Nucleotide and peptide sequence analyses were performed using theWisconsin Package (GCG, Genetics Computer Group, Madison, Wis.).Sequence analyses indicated that one clone, named rHY35a, contained anopen reading frame from the starting MET codon to the middle of apredicted seventh transmembrane domain. Because the high degree ofidentity of rHY35a to rGALR1 and rGALR2 indicated that it mightrepresent a fragment of a novel galanin receptor (referred to herein as“GALR3”), PCR primers directed to the amino terminus (forward primer)and first extracellular loop (reverse primer) of each of thecorresponding receptor cDNA were synthesized having the followingsequences:

rGALR1:

(forward primer): 5′-CCTCAGTGAAGGGAATGGGAGCGA-3′ (SEQ ID NO: 20);

(reverse primer): 5′-GTAGTGTATAAACTTGCAGATGAAGGC-3′ (SEQ ID NO: 21);

rGALR2:

(forward primer): 5′-ATGAATGGCTCCGGCAGCCAGGG-3′ (SEQ ID NO: 22);

(reverse primer): 5′-TTGCAGAGCAGCGAGCCGAACAC-3′ (SEQ ID NO: 23); and

rHY35a (i.e., rat GALR3):

(forward primer): 5′-GGCTGACATCCAGAACATTTCGCT-3′ (SEQ ID NO: 24);

(reverse primer): 5′-CAGATGTACCGTCTTGCACACGAA-3′ (SEQ ID NO: 25).

Polymerase Chain Reaction (PCR) of cDNA

Total RNA was prepared from RIN14B cells (ATCC No. CCL 89) by amodification of the guanidine thiocyanate method (Chirgwin et al.,1979). Poly A⁺ RNA was purified with a FastTrack kit (Invitrogen Corp.,San Diego, Calif.) and converted to single-stranded cDNA by randompriming using Superscript reverse transcriptase (BRL, Gaithersburg,Md.). An aliquot of the first strand cDNA was diluted (1:50) in a 50 μLPCR reaction mixture containing a combination of Taq and Pwo DNApolymerases in the buffer supplied by the manufacturer (for the ExpandLong Template PCR System, Boehringer Mannheim), and 300 nM each of theamino terminus and first extracellular loop rGALR3 (rHY35a) primersdescribed above. The PCR amplification reaction was performed under thefollowing conditions: 30 sec. at 94° C. and 1 min. 30 sec. at 68° C. for40 cycles, with a pre- and post-incubation of 5 min. at 95° C. and 2min. 30 sec. at 68° C., respectively. In order to control for theamplification of DNA (potentially carried over during the RNAextraction), control PCR reactions were run in parallel using RIN14B RNAprepared as above but without reverse transcriptase, and thus notconverted to cDNA. The PCR products were separated on a 1.0% agarose geland stained with ethidium bromide.

Construction and PCR Screening of a RIN14B Cell Line Plasmid Library

Total RNA was prepared from RIN14B cells by a modification of theguanidine thiocyanate method (Chirgwin et al., 1979). Poly A⁺ RNA waspurified with a FastTrack kit (Invitrogen Corp., San Diego, Calif.).Double stranded (ds) cDNA was synthesized from 4 Ag of poly A⁺ RNAaccording to Gubler and Hoffman (1983) with minor modifications. Theresulting cDNA was ligated to BstXI/EcoRI adaptors (Invitrogen Corp.)and the excess adaptors removed by exclusion column chromatography. Highmolecular weight fractions of size-selected ds-cDNA were ligated inPEXJ.BS (an Okayama and Berg expression vector) and electroporated in E.coli MC 1061 (Gene Pulser, Biorad). A total of 0.9×10⁶ independentclones with an insert mean size of 3.4 kb were generated. The librarywas plated on agar plates (Ampicillin selection) in 216 pools of ˜4,000independent clones. After 18 hours amplification, the bacteria from eachpool were scraped, resuspended in 4 mL of LB media, and 1.5 mL processedfor plasmid purification (Qiaprep, Qiagen, Inc., Chatsworth, Calif.).Aliquots of each bacterial pool were stored at −85° C. in 20% glycerol.

Glycerol stocks (2 μL) of the 216 primary pools for the RIN14B plasmidlibrary (designated “F”) were screened for rGALR3 by PCR using a forwardprimer from the third transmembrane domain of rGALR3(5′-CATCTGCTCATCTACCTCACCATG-3′ (SEQ ID NO: 26)) and a reverse primerfrom third intracellular loop of rGALR3 (5′-CATAGGAAACATAGCGTGCGTCCG-3′(SEQ ID NO: 27)). PCR was performed with the Expand Long Template PCRSystem, as described in the preceding section. Two positive pools, FLOSand F212, were subjected to further PCR analyses, using a forward primerto the amino terminus of rat GALR3 (described above) with a reverseprimer from the third intracellular loop (described above), as well asvector-anchored PCR (see below). These PCR analyses indicated that,although these clones were full-length, they were in the incorrectorientation in the expression vector (pEXJ.BS). Although these poolswere not further subdivided, the sequence missing from clone rHY35a(i.e., from the middle of TM7 through the stop codon) was determinedfrom the F105 clone, using vector-anchored PCR, as described below.

Vector-anchored PCR

To determine the orientation and size of the F105 cDNA insert (includingthe coding region, 5′ untranslated (UT) and 3′ UT regions) PCR wasconducted on glycerol stocks (2 μL) using combinations of vector-derivedprimers and gene-specific primers. The vector-derived forward primersequence was 5′-AAGCTTCTAGAGATCCCTCGACCTC-3′ (SEQ ID NO: 28); thereverse primer sequence was 5′-AGGCGCAGAACTGGTAGGTATGGAA-3′ (SEQ ID NO:29). The rGALR3-specific forward primer (in the sixth transmembranedomain) was 5′-GCTCATCCTCTGCTTCTGGTACG-3′ (SEQ ID NO: 30); the reverseprimer (in the first extracellular loop) was5′-CAGATGTACCGTCTTGCACACGAA-3′ (SEQ ID NO: 31). PCR was performed withthe Expand Long Template PCR System, as described above. The PCRproducts were separated on a 1.0% agarose gel and stained with ethidiumbromide.

A 1.2 kb vector-anchored PCR product generated from pool F105 using thesixth TM forward primer from rGALR3 and the vector-derived reverseprimer was isolated from a 1% TAE gel using a GENECLEAN III kit (BIO101, Vista, Calif.) and sequenced using AmpliTaq DNA Polymerase, FS(Perkin Elmer). Sequencing reactions were run on an ABI PRISM 377 DNASequencer and analyzed using the Wisconsin Package (GCG, GeneticsComputer Group, Madison, Wis.). The sequence information from thisvector-anchored PCR product corresponding to the predicted 3′ end of thenovel receptor gene indicated an overlap with rHY35a within the firsthalf of TM7. Downstream of this overlap was new sequence, consistentwith the second half of TM7 and the carboxy terminus, including anin-frame stop codon. Based on this newly acquired sequence, a reverseprimer, within the 3′UT, was synthesized (also containing a BamHI siteat the 5′ end, as indicated by the underline):5′-CGAGGATCCCAACTTTGCCTCTGCTTTTTGGTGG- 3′ (SEQ ID NO: 32).

Construction and PCR Screening of a Rat Hypothalamus Plasmid Library

Total RNA was prepared from rat hypothalami by a modification of theguanidine thiocyanate method (Chirgwin, 1979). Poly A⁺ RNA was purifiedusing a FastTrack kit (Invitrogen Corp., San Diego, Calif.). Doublestranded (ds) cDNA was synthesized from 6 μg of poly A⁺ RNA according toGubler and Hoffman (1983) with minor modifications. The resulting cDNAwas ligated to BstXI/EcoRI adaptors (Invitrogen Corp.) and the excessadaptors removed by exclusion column chromatography. High molecularweight fractions of size-selected ds-cDNA were ligated in pEXJ.T7 (anOkayama and Berg expression vector modified from pcEXV (Miller &Germain, 1986) to contain BstXI and other additional restriction sitesand a T7 promoter (Stratagene) and electroporated in E. coli MC 1061(Gene Pulser, Biorad). A total of 1.2×10⁶ independent clones with a meaninsert size of 3.2 kb were generated. The library (designated “K”) wasplated on agar plates (Ampicillin selection) in 373 primary pools of˜3,200 independent clones. After 18 hours amplification, the bacteriafrom each pool were scraped, resuspended in 4 mL of LB media and 0.75 mLprocessed for plasmid purification (QIAwell-96 ultra, Qiagen, Inc.,Chatsworth, Calif.). Aliquots of each bacterial pool were stored at −85°C. in 20% glycerol.

To screen the library for galanin binding, COS-7 cells were plated inslide chambers (Lab-Tek) in Dulbecco's modified Eagle medium (DMEM)supplemented with 10% calf serum, 100 U/mL of penicillin, 100 ug/mLstreptomycin, 2 mM L-glutamine (DMEM-C) and grown at 37° C. in ahumidified 5% CO₂ atmosphere for 24 hours before transfection. Cellswere transfected with miniprep DNA prepared from the primary pools(˜3,200 cfu/pool) of the rat hypothalamus cDNA library (“K” library)using a modification of the DEAE-dextran method (Warden & Thorne, 1968).Pools containing GALR1 and GALR2 were identified by PCR prior toscreening. The galanin binding assay was carried out after 48 hours.Cells were rinsed twice with phosphate-buffered saline (PBS) thenincubated with 2 nM ¹²⁵I-porcine galanin (NEN; specific activity ˜2200Ci/mmol) in 20mM HEPES-NaOH, pH 7.4, containing 1.26 mM CaCl₂, 0.81 mMMgSO₄, 0.44 mM KH₂PO₄, 5.4 mM KCl, 10 mM NaCl, 0.1% BSA, and 0.1%bacitracin for one hour at room temperature. After rinsing and fixationin 2.5% glutaraldehyde, slides were rinsed in PBS, air-dried, and dippedin photoemulsion (Kodak, NTB-2). After a 4 day exposure slides weredeveloped in Kodak D19 developer, fixed, and coverslipped (Aqua-Mount,Lerner Laboratories), then inspected for positive cells by brightfieldmicroscopy (Leitz Laborlux, 25× magnification).

PCR Screening of the Rat Hypothalamus cDNA Library

Glycerol stocks of the primary pools were combined into 40 superpools of10 primary pools and screened for rGALR3 by PCR using the same primersas described for the screening of the RIN14B plasmid library (seeabove). Primary pools from positive superpools (#3 and #17) wereinspected for galanin binding using the photoemulsion binding assaydescribed above and screened by PCR. The slide corresponding to poolK163 exhibited positive galanin binding. Pool K163 was then subjected toPCR with internal rGALR3 primers (TM3 forward primer and thirdintracellular loop reverse primer; described above), full-length primers(forward primer to the amino terminus, at the starting MET, and reverseprimer to the 3′ UT (containing a Bam HI site as above)) and with thevector and gene-specific primers (preceding section). These PCR analysesindicated that the primary pool K163 contained a full-length codingregion for rGALR3 in the correct orientation in the expression vector,pEXJ.T7. Pool K163 was further analyzed by PCR and shown to containGALR3 but not GALR1 nor GALR2, indicating that a novel galanin receptorcDNA was present in the pool and responsible for the galanin binding.The PCR primers used to confirm the absence of GALR1 and GALR2 in thepool are described below:

rGALR1:

Forward primer, KS-1311: 5′-CCTCAGTGAAGGGAATGGGAGCGA (SEQ ID NO: 33);

Reverse primer, KS-1447: 5′-CTTGCTTGTACGCCTTCCGGAAGT (SEQ ID NO: 34);

Human GALR1:

Forward primer, KS-1177: 5′-TGGGCAACAGCCTAGTGATCACCG-3′ (SEQ ID NO: 35);

Reverse primer, KS-1178: 5′-CTGCTCCCAGCAGAAGGTCTGGTT-3′ (SEQ ID NO: 36);

rGALR2:

Forward primer, KS-1543: 5′-ATGAATGGCTCCGGCAGCCAGGG-3′ (SEQ ID NO: 37);

Reverse primer, KS-1499: 5′-TTGGAGACCAGAGCGTAAACGATGG-3′ (SEQ ID NO:38).

The primary pool K163 was further subdivided and screened by PCR. Onepositive subpool, 163-30, was subdivided into 15 pools of 150 clones and15 pools of 500 clones and plated on agar plates (ampicillin selection).

Colonies were transferred to nitrocellulose membranes (Schleicher andSchuell, Keene, N.H.), denatured in 0.4 N NaOH, 1.5 M NaCl, renatured in1M Tris, 1.5 M NaCl, and UV cross-linked. Filters were hybridizedovernight at 400C in a buffer containing 50% formamide, 5×SSC, 7 mMTRIS, 1× Denhardt's solution and 25 μg/mL salmon sperm DNA (SigmaChemical Co.) and 10⁶ cpm/ml of overlapping 45-mer oligonucleotideprobes, filled-in using [α-³²P] dCTP and [α-³²P] dATP (800 Ci/mmol, NEN)and Klenow fragment of DNA polymerase (Boehringer Mannheim). Thefollowing probe sequence is directed to the amino terminus of rGALR3:

from the sense strand:5′-AGATGGCTGACATCCAGAACATTTCGCTGGACAGCCCAGGGAGCG-3′ (SEQ ID NO: 39);

from the antisense strand:5′-ATCACAGGCACTGCCACAGCCCCTACGCTCCCTGGGCTGTCCAGCG-3′ (SEQ ID NO: 40).

Filters were washed 2×15 minutes at room temperature in 2×SSC, 0.1% SDS,2×15 minutes at 50° C. in 0.1×SSC, 0.1% SDS, and exposed to BioMax MSX-ray film (Kodak) with corresponding Kodak intensifying screens for 6hours. One positive colony, 163-30-17, was amplified overnightseparately in 100 mL LB media and in 100 mL TB media and processed forplasmid purification using a standard alkaline lysis miniprep procedurefollowed by a PEG precipitation. Clone K163-30-17 was sequenced on bothstrands using AmpliTaq DNA Polymerase, FS (Perkin Elmer). Sequencingreactions were run on an ABI PRISM 377 DNA Sequencer and analyzed usingthe Wisconsin Package (GCG, Genetics Computer Group, Madison, Wis.).Clone K163-30-17 was given the designation K1086 and deposited with theATCC (Accession No. 97747).

Expression in COS-7 Cells for Whole Cell-slide Binding

To test the ability of K163-30-17 to confer galanin binding, COS-7 cellswere plated in slide chambers (Lab-Tek) in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% calf serum, 100 U/mL of penicillin,100 μg/mL streptomycin, 2mM L-glutamine (DMEM-c) and grown at 37° C. ina humidified 5% CO₂ atmosphere for 24 hours before transfection. Cellswere transfected with 1 μg of miniprep DNA from K163-30-17 or vectorcontrol using a modification of the DEAE-dextran method (Warden andThorne, 1968). 48 hours after transfection, cells were rinsed withphosphate-buffered saline (PBS) then incubated with 2 nM ¹²⁵I-porcinegalanin (NEN; specific activity ˜2200 Ci/mmol) in 20 mM HEPES-NaOH, pH7.4, containing 1.26 mM CaCl₂, 0.81 mM MgSO₄, 0.44 mM KH₂PO_(4, 5.4) mMKCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for one hour at roomtemperature. After rinsing and fixation in 2.5% glutaraldehyde, bindingof ¹²⁵I-galanin to cells on the slide was detected by autoradiographyusing BioMax MS film (Kodak) and an intensifying screen (Kodak). Thesignal from K163-30-17 transfected cells was compared with the signalfrom control vector transfected cells.

Cloning and Sequencing a Novel Human Galanin Receptor Fragment

A human placenta genomic library in X dash II (˜1.5×10⁶ totalrecombinants; Stratagene, LaJolla, Calif.) was screened using the sameset of overlapping oligonucleotide probes to TM regions 1-7 of rat GALR2and under the same hybridization and wash conditions as described forscreening the rat hypothalamus cDNA library (supra). Lambda phage cloneshybridizing with the probe were plaque purified and DNA was prepared forSouthern blot analysis (Southern, 1975; Sambrook et al., 1989).

One phage clone, plc21a, contained a 2.7 kb KpnI/EcoRI fragment whichhybridized with the rat GALR2 TM2 oligonucleotide probe and wassubsequently subcloned into a pUC vector. Nucleotide sequence analysiswas accomplished by sequencing both strands using cycle sequencing withAmpliTaq DNA Polymerase, FS (Perkin Elmer) and products run on theautomated fluorescent sequencer, the ABI Prism 377 Sequencer (ABI), andsequence analyses were performed using the Wisconsin Package (GCG,Genetics Computer Group, Madison, Wis.). DNA sequence analysis indicatedgreatest homology to the rat and human GALR1 and GALR2 genes. This clonewas a partial intron-containing gene fragment, encoding the starting METthrough to an intron in the second intracellular loop (i.e., TM 3/4loop).

Isolation of the Full-length Human GALR3 Receptor Gene

Sequence analyses of the cloned human genomic fragment indicated thepresence of a open reading frame from the starting MET codon down to apredicted intron in the second intracellular loop, with a nucleotideidentity of 88% (93% amino acid identity) with the rat GALR3 receptordescribed above (thus establishing this human genomic clone to be thehuman homologue of rat GALR3). Although this human genomic fragment wasnot full-length and contained an intron downstream of TM3, it isanticipated that a molecular biologist skilled in the art may isolatethe full-length, intronless version of the human GALR3 receptor geneusing standard molecular biology techniques and approaches such as thosebriefly described below:

Approach #1: Using PCR to screen commercial human cDNA phage librariesand in-house human cDNA plasmid libraries with primers to the humanGALR3 sequence (forward primer in amino terminus,5′-ATGGCTGATGCCCAGAACATTTCAC-3′ (SEQ ID NO: 41), and reverse primer infirst extracellular loop, 5′-AGCCAGGCATCCAGCGTGTAGAT-3′ (SEQ ID NO: 42),we have identified two commercial libraries and two proprietary plasmidlibraries that contain at least part of the human GALR3 gene, asfollows:

human fetal brain cDNA lambda ZAPII library (Stratagene);

human testis cDNA lambda ZAPII library (Stratagene);

human hypothalamus cDNA plasmid library (proprietary)-3 superpoolsidentified; and

human hippocampus cDNA plasmid library (proprietary)-3 superpoolsidentified.

One may determine whether these libraries contain full-length humanGALR3 by: (1) obtaining a purified clone from the lambda libraries byplaque-purification and then conducting hybridization screening usingprobes derived from rat GALR3 under reduced stringency, using standardprotocols and/or (2) using PCR to determine which pool of the humanplasmid library superpools contain the gene and then conductingvector-anchor PCR (as described in this patent) to determine if thesecDNAs are full-length. One problem which may arise with vector-anchoredPCR is a false-positive result, in which the PCR product size isconsistent with a full-length clone but the product actually contains anintron in the second intracellular loop. In this case, sequencing ofthis product would identify whether this product contains the intron oris intronless and full-length (also see Approach #2 below).

Approach #2: We have also determined that the phage clone containing METthru the intron in the second intracellular loop (i.e., TM3/4 loop),plc21a (see above), also contains at least part of the 3′ end of thegene, by using hybridization at reduced stringency with a probe to thethird extracellular loop (TM 6/7) derived from the rat GALR3 sequence:

5′-ACGGTCGCTTCGCCTTCAGCCCGGCCACCTACGCCTGTCGCCTGG-3′ (SEQ ID NO: 43).

Standard molecular biology techniques may be used to subclone either theentire intron-containing full-length human GALR3 (with confirmation thatit contains an in-frame stop codon) or subclone the part of the genefrom the intron in the second intracellular loop through the stop codon.This approach would permit one to utilize sequence around thetermination codon to design a primer which can be used with the primeraround the starting MET, to generate the full-length intronless humanGALR3 gene, using human cDNA as the target template. Alternatively, onemay use restriction enzymes to remove the intron and some adjacentcoding region from the intron-containing human GALR3 gene, and thenreplace the removed coding region by inserting a restrictionenzyme-digested PCR fragment amplified from a tissue shown to expressthe intronless form of the receptor.

Approach #3: As yet another alternative method, one could utilize 3′RACE to generate a PCR product from human cDNA expressing human GALR3(e.g., human brain), using a forward primer derived from known sequencebetween the starting MET thru the second intracellular loop (from thefragment already isolated). Such a PCR product could then be sequencedto confirm that it contains the rest of the coding region (without anintron), and then attached to the 5′ end of the molecule, using anoverlapping restriction site, or alternatively, its sequence could beused to design a reverse primer in the predicted 3′ UT region togenerate the full-length, intronless human GALR3 receptor gene with useof the primer at the starting MET codon and using human cDNA as targettemplate.

To this end, we have also determined that the phage clone containing METthrough the intron in the second intracellular loop (i.e. TM 3/4 loop),plc21a (see above), also contains at least part of the 3′ end of thegene, by using hybridization at reduced stringency with probes either tothe third extracellular loop (TM 6/7) or to TM 4, derived from the ratGALR3 sequence:

5′-ACGGTCGCTTCGCCTTCAGCCCGGCCACCTACGCCTGTCGCCTGG-3′ (SEQ ID NO: 44)

5′-GCGCAACGCGCGCGCCGCCGTGGGGCTCGTGTGGCTGCTGGCGGC-3′ (SEQ ID NO: 45).

Another clone, plc14a, which was essentially the same as plc21a (i.e.possessed the identical restriction map and hybridizing bands asplc21a), was further utilized by subcloning a 1.4 kb KpnI fragment whichsimilarly hybridized to the above probes. Since the phage clone, plc14a,also hybridized with a TM2/3 loop probe under high stringency, derivedfrom sequence data of human GALR3 5′ fragment (plc21a, see above),

5″-ATCTACACGCTGGATGCCTGGCTCTTTGGGGCCCTCGTCTGCAAG-3′ (SEQ ID NO: 46),

this 3′ fragment (e.g. plc14a) presumably corresponds to the 3′ end ofhuman GALR3 and is molecularly linked to the 5′ fragment (e.g. plc21a2.7 kb KpnI/EcoRI clone); however, an intron of unknown size separatesthe coding region, which is defined on the 5′ (2.7 kb KpnI/EcoRI plc21afragment) and 3′ (1.4 kb KpnIplc14a fragment) genomic pieces. Nucleotidesequence analysis was conducted on the 1.4 kb KpnI plc14a fragment, asdescribed above, and indicated greatest homology to the rat and humanGALR1 and GALR2 genes.

To obtain sequence information from the region defined by theintersection of these to exons as well as to prove that the 5′ and 3′fragments, putatively representing the entire full-length coding regionof human GALR3, are molecularly linked, we used a forwardoligonucleotide primer located on the 5′ fragment (within 2/3 loop)

5′-ATCTACACGCTGGATGCCCTGGCT-3′ (SEQ ID NO: 47) and a reverseoligonucleotide primer located on the 3′ fragment (within the predicted4/5 loop),

5′-CGTAGCGCACGGTGCCGTAGTA-3′ (SEQ ID NO: 48),

to amplify human brain and liver cDNA (corresponding to 5 ng of poly⁺RNA). The predicted =250 nts. PCR products were sequenced anddemonstrated that: (1) the sequences were identical between brain andliver cDNA, (2) the 5′ and 3′ genomic fragments are linked and representthe 5′ and 3′ fragments of the human GALR3 gene, and (3) the sequenceobtained defined the junction of the exon containing the starting METthrough the 3/4 loop (e.g., housed on the 2.7 kb KpnI/EcoRI plc21asubclone) and the exon containing the 3/4 loop through the predictedSTOP codon (e.g. housed on the 1.4 kb KpnI plc14a subclone). Thesequence of this junction demonstrated the presence of a KpnI site,which was utilized in the construction of the full-length gene.

The construction of the full-length human GALR3 gene first involved thegeneration of the 5′ end of the gene using PCR to synthetically create aKpnI site at the 3′ end of the PCR product. To this end, we designed aforward oligonucleotide primer located at the starting MET of the 5′fragment and added a consensus Kozak sequence as well as a BamHI site tobe used for subcloning:

5′-GATGGATCCGCCACCATGGCTGATGCCCAGAACATTTCAC-3′ (SEQ ID NO: 49),

and a reverse oligonucleotide primer, within the 3/4 loop, containing aKpnI site that generated the joint between the 5′ and 3′ KpnI fragment:

5′-GCAGGTACCTGTCCACGGAGACAGCAGC-3′ (SEQ ID NO: 50).

The addition of the KpnI site enabled the attachment of the 3′ KpnIfragment but preserved the sequence which was identified from humanbrain and liver cDNAs.

The forward and reverse primers were used to amplify the 2.7 kbKpnI/EcoRI5′ genomic-containing plasmid (plc21a) using PCR, as describedin a previous section but utilizing Expand High Fidelity PCR System(Boehringer Manniheim). The PCR product was isolated from a low meltinggel, purified by phenol extraction, digested with BamHI and KpnI andpurified further by phenol extraction. This BamHI/KpnI PCR product wassubcloned into BamHI/KpnI-digested expression vector, pEXJ, andsequenced. The sequence of the PCR product was identical to thatdetermined for the original genomic fragment. The subclone was thendigested with KpnI, treated with calf intestinal alkaline phosphatase,and ligated with the 1.4 KpnI 3′ genomic fragment. Correct orientationwas determined by both restriction mapping and sequencing. Therefore,the full-length human GALR3 construct contained =1.7 kb genomic insert,containing 1107 bp of coding region and =600 bp of 3′ non-coding region.

Northern Blots

Rat multiple tissue northern blots (rat MTN blot, Clontech, Palo Alto,Calif.), containing 2 μg poly A⁺ RNA, or northern blots containing 5 μgpoly⁺ RNA, either purchased from Clontech or purified from various ratperipheral tissues and brain regions, respectively, were similarlyhybridized at high stringency with a probe directed to theamino-terminus of rGalR3 (SEQ ID NO 39 and 40), according to themanufacturer's specifications. Probe was labeled as previously described(supra), using Klenow fragment of DNA polymerase, except [α-³²P] dCTPand [α-³²P] DATP (3000 Ci/mmol, NEN) were used. Northern blots werereprobed with a randomly-primed β-actin probe to assess quantities ofmRNA present in each lane.

Human brain multiple tissue northern blots (MTN brain blots II and III,Clontech, Palo Alto, Calif.) and human peripheral MTN blot (Clontech,Palo Alto, Calif.) carrying mRNA (2 μg) purified from various humanbrain areas and peripheral tissues, respectively, were hybridized athigh stringency with overlapping probes directed to the amino-terminusof hGALR3 5′ GATGGCTGATGCCCAGAACATTTCACTGGACAGCCCAGGGAGTGT 3′ (SEQ IDNO. 51) and 5′ GACCACAGGCACTGCCACGGCCCCCACACTCCCTGGGCTGTCCAG 3′ (SEQ IDNO. 52), according to the manufacturer's specifications.

RT-PCR Analyses of GALR3 mRNA

Tissues were homogenized and total RNA extracted using the guanidineisothiocyanate/CsCl cushion method. RNA was then treated with DNase toremove any contaminating genomic DNA and poly A⁺-selected usingFastTrack kit (Invitrogen), according to manufacturer's specifications.cDNA was prepared from mRNA with random hexanucleotide primers usingreverse transcriptase Superscript II (BRL, Gaithersburg, Md.). Firststrand cDNA (corresponding to ≈5 ng of poly A⁺ RNA) was amplified in a50 μL PCR reaction mixture with 300 nM of forward (directed to theamino-terminus: SEQ ID NO. 24) and reverse (directed to the thirdintracellular loop: SEQ ID NO. 27) primers, using the thermal cyclingprogram and conditions described above.

The PCR products were run on a 1.5% agarose gel and transferred tocharged nylon membranes (Zetaprobe GT, BioRad), and analyzed as Southernblots. GALR3 primers were screened for the absence of cross-reactivitywith the other galanin receptors. Filters were hybridized with aradiolabeled probe directed to the first intracellular loop,5′-TGCAGCCTGGCCCAAGTGCCTGGCAGGAGCCAAGCAGTACCACAG-3′ (Seq. I.D. No. 53),and washed under high stringency. Labeled PCR products were visualizedon X-ray film. Similar PCR and Southern blot analyses were conductedwith primers and probes directed to the housekeeping gene,glyceraldehyde phosphate dehydrogenase (G3PDH; Clontech, Palo Alto,Calif.), to normalize the amount of cDNA used from the differenttissues.

Production of Recombinant Baculovirus

The coding region of GALR3 may be subcloned into pBlueBacIII intoexisting restriction sites, or sites engineered into sequences 5′ and 3′to the coding region of GALR3, for example, a 5′ EcoRI site and a 3′EcoRI site. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of GALR3 construct may be co-transfected into 2×10⁶ Spodopterafrugiperda insect Sf9 cells by the calcium phosphate co-precipitationmethod, as outlined in by Pharmingen (in “Baculovirus Expression VectorSystem: Procedures and Methods Manual”). The cells then are incubatedfor 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Cell Culture

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/mL penicillin/100 μg/mL streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days. Human embryonic kidney 293 cells are grown on 150 mm plates inD-MEM with supplements (minimal essential medium) with Hanks' salts andsupplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mL streptomycin)at 37° C., 5% CO₂. Stock plates of 293 cells are trypsinized and split1:6 every 3-4 days. Mouse fibroblast LM(tk-) cells are grown on 150 mmplates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk-) cells aretrypsinized and split 1:10 every 3-4 days.

LM(tk-) cells stably transfected with the GALR3 receptor may beroutinely converted from an adherent monolayer to a viable suspension.Adherent cells are harvested with. trypsin at the point of confluence,resuspended in a minimal volume of complete DMEM for a cell count, andfurther diluted to a concentration of 10⁶ cells/mL in suspension media(10% bovine calf serum, 10% 10× Medium 199 (Gibco), 9 mM NaHCO₃, 25 mMglucose, 2 mM L-glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin, and 0.05% methyl cellulose). Cell suspensions aremaintained in a shaking incubator at 37° C., 5% CO₂ for 24 hours.Membranes harvested from cells grown in this manner may be stored aslarge, uniform batches in liquid nitrogen. Alternatively, cells may bereturned to adherent cell culture in complete DMEM by distribution into96-well microtiter plates coated with poly-D-lysine (0.01 mg/mL)followed by incubation at 37° C., 5% CO₂ for 24 hours. Cells prepared inthis manner generally yield a robust and reliable response in cAMPradio-immunoassays as further described hereinbelow.

Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days. Chinese hamster ovary (CHO)cells were grown on 150 mm plates in HAM's F-12 medium with supplements(10% bovine calf serum, 4 mM L-glutamine and 100 units/mL penicillin/100ug/ml streptomycin) at 37° C., 5% CO₂. Stock plates of CHO cells weretrypsinized and split 1:8 every 3-4 days.

Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells are grown on 150 mm tissue culturedishes in Ex-Cell 400′ medium supplemented with L-Glutamine, also at270C, no CO₂.

Transfection

All receptor subtypes studied may be transiently transfected into COS-7cells by the DEAE-dextran method, using 1 μg of DNA/10⁶ cells (Cullen,1987). In addition, Schneider 2 Drosophila cells may be cotransfectedwith vectors containing the receptor gene, under control of a promoterwhich is active in insect cells, and a selectable resistance gene, eg.,the G418 resistant neomycin gene, for expression of the galaninreceptor.

Stable Transfection

The GALR3 receptor may be co-transfected with a G-418 resistant geneinto the human embryonic kidney 293 cell line by a calcium phosphatetransfection method (Cullen, 1987). Stably transfected cells areselected with G-418. GALR3 receptors may be similarly transfected intomouse fibroblast LM(tk-) cells, Chinese hamster ovary (CHO) cells andNIH-3T3 cells, or other suitable host cells.

GALR1 receptors were expressed in cells using methods well-known in theart.

Radioligand Binding Assays

Transfected cells from culture flasks are scraped into of 20 mMTris-HCl₁, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysatesare centrifuged at 1000 rpm for 5 min. at 4° C., and the supernatantcentrifuged at 30,000×g for 20 min. at 4° C. The pellet is suspended inbinding buffer (50 mM Tris-HCl, 5 mM MgSO₄, 1 mM EDTA at pH 7.5supplemented with 0.1% BSA, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and10 μg/ml phosphoramidon). Optimal membrane suspension dilutions, definedas the protein concentration required to bind less than 10% of the addedradioligand, are added to 96-well polpropylene microtiter platescontaining ¹²⁵I-labeled peptide, non-labeled peptides and binding bufferto a final volume of 250 μl. In equilibrium saturation binding assaysmembrane preparations may be incubated in the presence of increasingconcentrations (e.g., 0.1 nM to 4 nM) of [¹²⁵I] porcine galanin(specific activity about 2200 Ci/mmol). The binding affinities of thedifferent galanin analogs may be determined in equilibrium competitionbinding assays, using 0.1-0.5 nM [¹²⁵I] porcine galanin in the presenceof e.g., twelve different concentrations of the displacing ligands.Binding reaction mixtures are incubated for 1 hr at 30° C., and thereaction stopped by filtration through GF/B filters treated with 0.5%polyethyleneimine, using a cell harvester. Radioactivity may be measuredby scintillation counting and the data analyzed by a computerizednon-linear regression program. Non-specific binding may be defined asthe amount of radioactivity remaining after incubation of membraneprotein in the presence of 100 nM of unlabeled porcine galanin. Proteinconcentration may be measured by the Bradford method using Bio-RadReagent, with bovine serum albumin as a standard.

The binding assays used to generate the data shown in Table 3 wereconducted as described above, with certain modifications. Assays wereconducted at room temperature for 120 minutes, and leupeptin, aprotoninand phosphoramidon were omitted from the rat GALR3 assay, whilebacitracin was added to 0.1%. In addition, nonspecific binding wasdefined in the presence of 1 μM porcine galanin.

Functional Assays

Cyclic AMP (cAMP) formation

The receptor-mediated inhibition of cyclic AMP (cAMP) formation may beassayed in LM(tk-) cells expressing the galanin receptors. Cells areplated in 96-well plates and incubated in Dulbecco's phosphate bufferedsaline (PBS) supplemented with 10 mM HEPES, 5 mM theophylline, 2 μg/mlaprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon for 20 minat 37° C., in 5% CO₂. Galanin or the test compounds are added andincubated for an additional 10 min at 37° C. The medium is thenaspirated and the reaction stopped by the addition of 100 mM HCl. Theplates are stored at 4° C. for 15 min, and the cAMP content in thestopping solution measured by radioimmunoassay. Radioactivity may bequantified using a gamma counter equipped with data reduction software.

Arachidonic Acid Release

CHO cells stably transfected with the rat GALR3 receptor are seeded into96 well plates and grown for 3 days in HAM's F-12 with supplements.³H-arachidonic acid (specific activity =0.75 uCi/ml) is delivered as a100 uL aliquot to each well and samples were incubated at 37° C., 5% CO₂for 18 hours. The labeled cells are washed three times with 200 uL HAM'sF-12. The wells are then filled with medium (200 uL) and the assay isinitiated with the addition of peptides or buffer (22 uL). Cells areincubated for 30 min at 37° C., 5% CO₂. Supernatants are transferred toa microtiter plate and evaporated to dryness at 75° C. in a vacuum oven.Samples are then dissolved and resuspended in 25 uL distilled water.Scintillant (300 uL) is added to each well and samples are counted for³H in a Trilux plate reader. Data are analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

Intracellular Calcium Mobilization

The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al. 1991) Stably transfected cells are seeded onto a 35 mmculture dish containing a glass coverslip insert. Cells are washed withHBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40 min. Afterwashing with HBS to remove the Fura-2/AM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

Phosphoinositide Metabolism

LM(tk-) cells stably expressing the rat GALR3 receptor cDNA are platedin 96-well plates and grown to confluence. The day before the assay thegrowth medium is changed to 100 μl of medium containing 1% serum and 0.5μCi [³H] myo-inositol, and the plates are incubated overnight in a CO₂incubator (5% CO₂ at 37° C.). Alternatively, arachidonic acid releasemay be measured if [³H] arachidonic acid is substituted for the [³H]myo-inositol. Immediately before the assay, the medium is removed andreplaced by 200 μL of PBS containing 10 mM LiCl, and the cells areequilibrated with the new medium for 20 min. During this interval cellsare also equilibrated with the antagonist, added as a 10 μL aliquot of a20-fold concentrated solution in PBS. The [³H] inositol-phosphatesaccumulation from inositol phospholipid metabolism may be started byadding 10 μL of a solution containing the agonist. To the first well 10μL may be added to measure basal accumulation, and 11 differentconcentrations of agonist are assayed in the following 11 wells of eachplate row. All assays are performed in duplicate by repeating the sameadditions in two consecutive plate rows. The plates are incubated in aCO₂ incubator for 1 hr. The reaction may be terminated by adding 15 μlof 50% v/v trichloroacetic acid (TCA), followed by a 40 min. incubationat 4° C. After neutralizing TCA with 40 μl of 1M Tris, the content ofthe wells may be transferred to a Multiscreen HV filter plate(Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). Thefilter plates are prepared adding 200 μL of Dowex AG1-X8 suspension (50%v/v, water: resin) to each well. The filter plates are placed on avacuum manifold to wash or elute the resin bed. Each well is washed 2times with 200 μL of water, followed by 2×200 μL of 5 mM sodiumtetraborate/60 mM ammonium formate. The [³H] IPs are eluted into empty96-well plates with 200 μl of 1.2 M ammonium formate/0.1 formic acid.The content of the wells is added to 3 mls of scintillation cocktail,and the radioactivity is determined by liquid scintillation counting.

The binding and functional assays described herein may also be performedusing GALR1 and GALR2 receptors. The GALRL receptors are well-known inthe art and may be prepared and transfected into cells (transiently andstably) using standard methods. Applicants have isolated and cloned therat and human GALR2 receptors, and have deposited several plasmidsexpressing GALR2 receptors, as well as cell lines stably expressing therat GALR2 receptor. Plasmids expressing GALR2 receptors may betransiently or stably transfected into cell using methods well-known inthe art, examples of which are provided herein. The rat GALR2 receptormay be expressed using plasmid K985 (ATCC Accession No. 97426, depositedJan. 24, 1996), or using plasmid K1045 (ATCC Accession No. 97778,deposited Oct. 30, 1996). Plasmid K1045 comprises an intronlessconstruct encoding the rat GALR2 receptor. Cell lines stably expressingthe rat GALR2 receptor have also been prepared, for example, the LM(tk-)cell lines L-rGALR2-8 (ATCC Accession No. CRL-12074, deposited Mar. 28,1996) and L-rGALR2I-4 (ATCC Accession No. CRL-12223, deposited Oct. 30,1996). L-rGALR2I-4 comprises an intronless construct expressing the ratGALR2 receptor. The CHO cell line C-rGalR2-79 (ATCC Accession No.CRL-12262, deposited Jan. 15, 1997) also stably expresses the rat GALR2receptor. The human GALR2 receptor may be expressed using plasmid BO29(ATCC Accession No. 97735, deposited Sep. 25, 1996) or plasmid BO39(ATCC Accession No. 97851, deposited Jan. 15, 1997). Plasmid B039comprises an intronless construct encoding the human GALR2 receptor.

The plasmids and cell lines described above were deposited with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure.

It is to be understood that the cell lines described herein are merelyillustrative of the methods used to evaluate the binding and function ofthe galanin receptors of the present invention, and that other suitablecells may be used in the assays described herein.

Methods for Recording Currents in Xenopus oocytes

Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) are anesthetized in0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) anda portion of ovary is removed using aseptic technique (Quick and Lester,1994). Oocytes are defolliculated using 2 mg/ml collagenase (WorthingtonBiochemical Corp., Freehold, N.J.) in a solution containing 87.5 mMNaCl, 2 mM KCl, 2 mM MgCl₂ and 5 mM HEPES, pH 7.5. Oocytes are injected(Nanoject, Drummond Scientific, Broomall, Pa.) with 50 nL of rat GalR3mRNA. Other oocytes are injected with a mixture of GalR3 mRNA and mRNAencoding the genes for G-protein-activated inward rectifiers (GIRK1 andGIRK4). Genes encoding GIRK1 and GIRK4 are obtained using conventionalPCR-based cloning techniques based on published sequences (Kubo et al.,1993; Dascal et al., 1993; Krapivinsky et al., 1995). RNAs are preparedfrom separate DNA plasmids containing the complete coding regions ofGalR3, GIRK1 and GIRK4. Plasmids are linearized and transcribed usingthe T7 polymerase (“Message Machine”, Ambion). Alternatively, mRNA maybe translated from a template generated by PCR, incorporating a T7promoter. After injection of mRNA, oocytes are incubated at 16° on arotating platform for 3-8 days. Dual electrode voltage clamp(“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) is performedusing 3 M KCl-filled glass microelectrodes having resistances of 1-3Mohms. Unless otherwise specified, oocytes are voltage clamped at aholding potential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mM HEPES, pH 7.5 (“ND96”), or, in thecase of oocytes expressing GIRK1 and GIRK4, elevated K⁺ containing 96 mMKCl, 2 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mM HEPES, pH 7.5 (“hK”).Drugs are applied by switching from a series of gravity fed perfusionlines.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activationof the phospholipase C (PLC) pathway is assayed by applying 1 μM galaninin ND96 solution to oocytes previously injected with mRNA for the GalR3receptor and observing inward currents at a holding potential of −80 mV.The appearance of currents that reverse at −25 mV and display otherproperties of the Ca⁺⁺-activated Cl⁻ channel is indicative of GalR3receptor-activation of PLC and release of IP3 and intracellular Ca⁺⁺.Subsequently, measurement of inwardly rectifying K⁺ channel (GIRK)activity is monitored in oocytes that have been co-injected with mRNAsencoding GALR3, GIRK1 and GIRK4. These two GIRK gene productsco-assemble to form a G-protein activated potassium channel known to beactivated by a number of GPCRs that couple to G_(i) or G_(o) (Kubo etal., 1993; Dascal et al., 1993). Oocytes expressing GalR3 plus the twoGIRK subunits are tested for galanin responsivity using 1 μM galanin andmeasuring K⁺ currents in elevated K⁺ solution (hK). Activation ofinwardly rectifying currents that are sensitive to 100 μM Ba⁺⁺ signifiesGALR3 coupling to a G_(i) or G_(o) pathway in the oocytes.

Oocytes were isolated as described above, except that 3 mg/mLcollagenase was used to defolliculate the oocytee. Genes encodingG-protein inwardly rectifying K⁺ channels 1 and 4 (GIRK1 and GIRK4) wereobtained by PCR using the published sequences (Kubo et al., 1993; Dascalet al., 1993; Krapivinsky et al., 1995b) to derive appropriate 5′ and 3′primers. Human heart cDNA was used as template together with the primers5′-CGCGGATCCATTATGTCTGCACTCCGAAGGAAATTTG-3′ (SEQ ID NO. 54) and5′-CGCGAATTCTTATGTGAAGCGATCAGAGTTCATTTTTC -3′ (SEQ ID NO. 55) for GIRK1and 5′-GCGGGATCCGCTATGGCTGGTGATTCTAGGAATG-3′ (SEQ ID NO. 56) and 5′-CCGGAATTCCCCTCACACCGAGCCCCTGG-3′ (SEQ ID NO. 57) for GIRK4. In eachprimer pair, the upstream primer contained a BamHI site and thedownstream primer contained an EcoRI site to facilitate cloning of thePCR product into pcDNA1-Amp (Invitrogen). The transcription template forhGalR3 was obtained similarly by PCR using the cloned cDNA incombination with primers5′-CCAAGCTTCTAATACGACTCACTATAGGGCCACCATGGCTGATGCCCAGA-3′ (SEQ ID NO. 58)and 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGGG TTTATTCCGGTCCTCG-3′(SEQ ID NO. 59). mRNAs were transcribed using the T7 polymerase(“Message Machine”, Ambion). Each oocyte received 2 ng each of GIRK1 andGIRK4 mRNA in combination with 25 ng of GalR3 mRNA. In other experimentsoocytes received injections of mRNAs encoding the human alA adrenergicreceptor, rGalR1 or rGalR2 galanin receptors (Forray et al., 1994;Parker et. al., 1995) with or without GIRKs 1 and 4. After injection ofmRNAs, oocytes were incubated at 170 for 3-8 days.

Dual electrode voltage clamp (“GeneClamp”, Axon Instruments Inc., FosterCity, Calif.) was performed as described above, with the followingmodifications: during recordings, oocytes were bathed in continuouslyflowing (1-3 mL/min) ND96 medium or, in the case of oocytes expressingGIRKs 1 and 4, elevated K⁺ containing 48 mM KCl, 49 mM NaCl, 2 mM CaCl₂,2 mM MgCl₂, and 5 mM HEPES, pH 7.5 (1/2 hK). Drugs were applied eitherby local perfusion from a 10 μl glass capillary tube fixed at a distanceof 0.5 mm from the oocyte, or for calculation of steady-state EC₅₀s, byswitching from a series of gravity fed perfusion lines. Experiments werecarried out at room temperature. All values are expressed as mean+standard error of the mean.

Tissue Preparation for Neuroanatomical Studies

Male Sprague-Dawley rats (Charles River, Wilmington, Mass.) aredecapitated and the brains rapidly removed and frozen in isopentane.Coronal sections may be cut at 11 μm on a cryostat and thaw-mounted ontopoly-L-lysine coated slides and stored at −80° C. until use. Prior tohybridization, tissues are fixed in 4% paraformaldehyde, treated with 5mM dithiothreitol, acetylated in 0.1 M triethanolamine containing 0.25%acetic anhydride, delipidated with chloroform, and dehydrated in gradedethanols.

Probes

oligonucleotide probes employed to characterize the distribution of therat GALR3 receptor mRNA may be synthesized, for example, on a MilliporeExpedite 8909 Nucleic Acid Synthesis System. The probes are thenlyophilized, reconstituted in sterile water, and purified on a 12%polyacrylamide denaturing gel. The purified probes are againreconstituted to a concentration of 100 ng/μL, and stored at −20° C.Probe sequences may include DNA or RNA which is complementary to themRNA which encodes the GALR3 receptor.

In Situ Hybridization

Probes are 3′-end labeled with ³⁵S-dATP (1200 Ci/mmol, New EnglandNuclear, Boston, Mass.) to a specific activity of about 10⁹ dpm/μg usingterminal deoxynucleotidyl transferase (Pharmacia). The radiolabeledprobes are purified on Biospin 6 chromatography columns (Bio-Rad;Richmond, Calif.), and diluted in hybridization buffer to aconcentration of 1.5×10⁴ cpm/μL. The hybridization buffer consists of50% formamide, 4× sodium citrate buffer (1×SSC =0.15 M NaCl and 0.015 Msodium citrate), 1× Denhardt's solution (0.2% polyvinylpyrrolidine, 0.2%Ficoll, 0.2% bovine serum albumin), 50 mM dithiothreitol, 0.5 mg/mlsalmon sperm DNA, 0.5 mg/ml yeast tRNA, and 10% dextran sulfate. Aboutone hundred μL of the diluted radiolabeled probe is applied to eachsection, which is then covered with a Parafilm coverslip. Hybridizationis carried out overnight in humid chambers at 40 to 55° C. The followingday the sections are washed in two changes of 2×SSC for one hour at roomtemperature, in 2×SSC for 30 min at 50-60° C., and finally in 0.1×SSCfor 30 min at room temperature. Tissues are dehydrated in gradedethanols and apposed to Kodak XAR-5 film for 3 days to 3 weeks at −20°C., then dipped in Kodak NTB3 autoradiography emulsion diluted 1:1 with0.2% glycerol water. After exposure at 4° C. for 2 to 8 weeks, theslides are developed in Kodak D-19 developer, fixed, and counterstainedwith cresyl violet.

Solution Hybridization/Ribonuclease Protection Assay

For solution hybridization 2-15 μg of total RNA isolated from tissuesmay be used. Sense RNA synthesized using the full-length coding sequenceof the rGalR2 is used to characterize specific hybridization. Negativecontrols may consist of 30 μg transfer RNA (tRNA) or no tissue blanks.Samples are placed in 1.5-ml microfuge tubes and vacuum dried.Hybridization buffer (40 μl of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mMEDTA, in 80% formamide) containing 0.25-1.0×10⁶ counts of each probe isadded to each tube. Samples are heated at 90° C. for 15 min, after whichthe temperature is lowered to 45° C. for hybridization.

After hybridization for 14-18 hr, the RNA/probe mixtures are digestedwith RNAse A (Sigma) and RNAse T1 (Bethesda Research Labs, Gaithersburg,Md.). A mixture of 2.0 μg RNAse A and 1000 units of RNAse T1 in a buffercontaining 330 mM NaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) isadded to each sample and incubated for 90 min at room temperature. Afterdigestion with RNAses, 20 μl of 10% SDS and 50 μg proteinase K are addedto each tube and incubated at 37° C. for 15 min. Samples are thenextracted with phenol/chloroform:isoamyl alcohol and precipitated in 2volumes of ethanol for 1 hr at −70° C. tRNA is added to each tube (30mg) as a carrier to facilitate precipitation. Following precipitation,samples are centrifuged, washed with cold 70% ethanol, and vacuum dried.Samples are dissolved in formamide loading buffer and size-fractionatedon a urea/acrylamide sequencing gel (7.6 M urea, 6% acrylamide inTris-borate-EDTA). Gels are dried and apposed to Kodak XAR-5 x-ray film.

In Vivo Methods

The effects of galanin, galanin derivatives, and related peptides andcompounds may be evaluated by intracerebroventricular (i.c.v.) injectionof the peptide or compound followed by measurement of food intake in theanimal. Measurement of food intake was performed for hours afterinjection, but other protocols may also be used. Saline was injected asa control, but it is understood that other vehicles may be required ascontrols for some peptides and compounds. In order to determine whethera compound is a GALR3 antagonist, food intake in rats may be stimulatedby administration of (for example) a galanin receptor agonist through anintracerebroventricular (i.c.v.) cannula. A preferred anatomic locationfor injection is the hypothalamus, in particular, the paraventricularnucleus. Methods of cannulation and food intake measurements arewell-known in the art, as are i.c.v. modes of administration (Kyrkouliet al., 1990, Ogren et al., 1992). To determine whether a compoundreduces agonist-stimulated food intake, the compound may be administeredeither simultaneously with the peptide, or separately, either throughcannula, or by subcutaneous, intramuscular, or intraperitonealinjection, or more preferably, orally.

Materials

Cell culture media and supplements are from Specialty Media (Lavallette,N.J.). Cell culture plates (150 mm and 96-well microtiter) are fromCorning (Corning, N.Y.). Sf9, Sf21, and High Five insect cells, as wellas the baculovirus transfer plasmid, pBlueBacIII™, are purchased fromInvitrogen (San Diego, Calif.). TMN-FH insect medium complemented with10% fetal calf serum, and the baculovirus DNA, BaculoGold™, is obtainedfrom Pharmingen (San Diego, Calif.). Ex-Cell 400™ medium withL-Glutamine is purchased from JRH Scientific. Polypropylene 96-wellmicrotiter plates are from Co-star (Cambridge, Mass.). All radioligandsare from New England Nuclear (Boston, Mass.)

Galanin and related peptide analogs were either from Bachem Calif.(Torrance, Calif.), Peninsula (Belmont, Calif.); or were synthesized bycustom order from Chiron Mimotopes Peptide Systems (San Diego, Calif.).

Bio-Rad Reagent was from Bio-Rad (Hercules, Calif.). Bovine serumalbumin (ultra-fat free, A-7511) was from Sigma (St. Louis. Mo.). Allother materials were reagent grade.

EXPERIMENTAL RESULTS Isolation of a Partial GALR3 cDNA from RatHypothalamus

In order to clone additional members of the galanin receptor family, ahomology cloning strategy based on the potential presence of multiplegalanin receptors in hypothalamus was designed. Although recent evidenceindicated that GALR1 and GALR2 receptor mRNAs were present in rathypothalamus (Gustafson et al., 1996; Parker et al., 1995), not allaspects of the cloned GALR1 and GALR2 pharmacological profiles matchthat observed for galanin-mediated feeding (Crawley et al., 1993). Theseresults suggested that the regulation of galanin-induced feeding may notbe explained by the presence of only GALR1 or GALR2 (or both) in the rathypothalamus.

In order to attempt to isolate additional galanin receptors, a rathypothalamus cDNA phage library was screened, under reduced stringencyconditions, with oligonucleotide probes directed to the transmembraneregions of the rat GALR2 neuropeptide receptor gene. Fivepositively-hybridizing clones were isolated, plaque-purified andcharacterized by Southern blot analysis and sequencing. One clone,rHY35a, contained a 3.5 kb insert (consisting of a 1.0 kb, 0.2 kb, and2.3 kb EcoRI fragments), which hybridized with the second transmembranedomain oligonucleotide probe of rat GALR2. DNA sequence analysisindicated greatest homology to the published rat GALR1 gene (Burgevin,et al., 1995) and the novel rat GALR2 receptor gene we have recentlyidentified. This clone was a partial intronless gere fragment,containing an open reading frame and encoding a predicted starting METthrough the middle of the predicted seventh transmembrane domain, with≈150 nucleotides of 5′ UT. Hydropathy analysis of the predictedtranslated protein is consistent with a putative topography of at leastsix transmembrane domains (the predicted sequence ended in the middle ofTM7), indicative of the G protein-coupled receptor family. This genefragment exhibited 52% and 66% nucleotide identity and 37% and 60% aminoacid identity to the rat GALR1 and rat GALR2 receptors, respectively.Furthermore, PCR primers directed to the amino terminus (forward primer)and first extracellular loop (reverse primer) of each of thecorresponding receptor genes, rGALR1 and rGALR2, were unable to amplifythis clone, whereas primers directed to this clone resulted in thecorrect size PCR product. The putative six (or seven) transmembranetopography and the high degree of identity to rat GALR1 and GALR2suggested that this cDNA represented a partial gene fragment of a novelgalanin-like receptor gene, referred to herein as GALR3.

In order to obtain the full-length gene, PCR on cDNA derived from theRIN14B cell line, using internal primers directed to TM3 and thirdintracellular loop of rat GALR3 was first conducted. It was hypothesizedthat since previous data indicated that this cell line expressed bothGALR1 and GALR2, it may also contain further subtypes. PCR analysesrevealed the presence of at least a portion of GALR3 in cDNA from RIN14Bcells; the absence of reverse transcriptase did not result in PCRamplification, indicating the ability to amplify RIN14B cDNA was due toauthentic GALR3 mRNA and not any contaminating genomic DNA in the RNAsource.

To isolate a cDNA molecule from RIN14B which expresses GALR3, a RIN14Bplasmid library was screened by PCR (using internal primers) and twopools, F105 and F212, were identified which contained a PCR product ofthe correct size. To determine if the insert was in the correctorientation for expression and to determine the size of the cDNA insert(including the coding region, 5′UT and 3′UT), vector-anchored PCR wasconducted on each pool. The PCR analyses suggested that both poolscontained full-length GALR3 but in the incorrect orientation and thuswould be predicted not to express the GALR3 receptor. Examination ofslides of COS-7 cells which had been transfected with DNA from each ofthese pools and subsequently bound with radioligand confirmed theabsence of binding of radiolabeled galanin, presumably due to itsincorrect orientation.

Although the full-length clone of rat GALR3 in the correct orientationfrom the RIN14B plasmid library was not obtained, it was reasoned thatthe sequence of the missing 3′ end (i.e., from the middle of TM7 throughthe stop codon) could be obtained by sequencing the vector-anchored PCRproduct corresponding to the 3′ end of the molecule. An ≈1.2 kb PCRproduct from a vector-anchored amplification of bacterial glycerol stockof the F105 pool was obtained, using a vector-derived reverse primer anda rGALR3-specific forward primer from TM6. This PCR product wassequenced with the gene-specific primer to reveal an overlap within TM7with the sequence known from rHY35a. In addition, further sequence wasobtained representing an open reading frame corresponding to the missingsecond half of TM7 and the carboxy terminus. The sequence obtainedshowed an overall 47% nucleotide identity to rGalR2, and a 62%nucleotide identity to rGalR2 from the third extracellular domain to the5′ end of the COOH terminus, confirming the existence of an open readingframe from a starting MET through a stop codon, with the presence ofseven putative transmembrane domains. Furthermore, this sequencepermitted us to design an oligonucleotide primer in the 3′ UT whichcould serve as a diagnostic tool for determination of full-lengthcharacterization of additional pools of DNA (see below).

Since the most convenient method to obtain the full-length rGALR3 clonein the correct orientation in an expression vector is to locate afull-length clone in preexisting libraries, and it was known that thisgene was expressed in rat hypothalamus, we screened a rat hypothalamusplasmid library (“K”) by PCR. Two superpools from the K library (#3 and#17) were identified as containing rGALR3. A primary pool, K163 (fromsuperpool #17), was identified to be positive and full-length usinginternal and full-length PCR primers, and vector-anchor primers wereused to determine the orientation. These data were consistent withprimary pool K163 (made up of 3200 primary clones), containingfull-length rGALR3 in the correct orientation in the expression vector,pEXJ.T7. Furthermore, this pool failed to amplify with GALR1- andGALR2-specific primers and yet exhibited galanin binding when DNA fromthis pool was used to transfect COS cells and tested for radiolabeledgalanin binding. These data suggested that a pool from a rathypothalamus plasmid cDNA library which contains the novel sequenceinitially identified from rat hypothalamus as a galanin-like receptorhad been identified, which, in addition, exhibits galanin binding,thereby identifying the pool as containing a novel galanin receptor,referred to herein as GALR3, or more specifically, rGALR3.

The pool K163 was then sib selected through one round by PCR and asecond round by colony hybridization, using a probe directed to theamino terminus of the sequence from rHY35a, resulting in the isolationof a single clone (i.e., a bacterial colony containing rat GALR3),called K163-30-17, representing the full-length rat GALR3 in the correctorientation. The rGALR3 recombinant bacterial colony was grown up inbroth with ampicillin and DT4A extracted. Restriction enzyme digestionsuggested a 2.1 kb insert, consistent with the clone comprising thefull-length coding region.

Furthermore, sequence analysis on K163-30-17 DNA (plasmid K1086)confirmed that it contained a full-length coding region in the correctorientation for expression.

Northern Blot Analyses of GALR3 mRNA

To define the size and distribution of the mRNA encoding GALR3, Northernblot analyses of poly A⁺ RNA from various rat tissues and brain regionswas carried out. A radiolabeled 70-mer oligonucleotide probe directed tothe amino terminus of the rat GALR3 coding region was used as ahybridization probe under high stringency. This probe failed tocross-hybridize with either the GALR1 or GALR2 genes under similarhybrization conditions, demonstrating its specificity for GALR3receptor. A single transcript of ≈3.3 kb is detected after a 5 dayexposure of the autoradiogram at −80° C. using Kodak Biomax MS film witha Biomax MS intensifying screen. GALR3 mRNA was not detected by Northernanalysis in the brain nor in various regions of the brain (see Table 1).Among various rat tissues, the GALR3 transcript had a restricteddistribution; GALR3 mRNA was predominantly observed in kidney with afaint signal detected in liver (see Table 1). This distribution was thesame upon a longer exposure of the autoradiogram (14 days). Northernblots were reprobed with G3PDH probe to assess whether similar amountsof mRNA were present in each lane.

Northern blot analyses of poly A+ RNA from various human brain regionsand peripheral tissues were carried out with a radiolabeled 70-meroligonucleotide probe directed to the amino terminus of the human GALR3coding region under high stringency. As demonstrated for thecorresponding rat probe, this human probe failed to cross-hybridize witheither the human GALR1 or GALR2 genes under similar hybridizationconditions, demonstrating its specificity for human GALR3 receptor. Notranscript was observed even after 14 day exposure of the autoradiogramin any of the human brain regions or peripheral tissues, by Northernblot analyses. The regions of the brain and periphery included in thisanalysis, as contained in the MTN blots from Clontech, included:amygdala, caudate nucleus, corpus callosum, hippocampus, total brain,substantial nigra, subthalamic brain, thalamus nucleus, cerebellum,cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe,temporal lobe, putamen, heart, total brain, placenta, lung, liver,skeletal muscle, kidney, and pancreas.

Reverse-transcription PCR of GALR3 mRNA

Amplification of cDNA derived from mRNA of various rat peripheral andbrain regions demonstrated the presence of GALR3 mRNA in various regionsof the brain, including hypothalamus (see Table 2), as well as severalperipheral tissues tested, such as pancreas and liver. It wasanticipated that we would identify GALR3 mRNA in hypothalamus since thegene was cloned from this region of the brain (supra). Therapeuticindications implied from localization of GALR3 mRNA for several of theseregions are also indicated in Table 2.

TABLE 1 Northern blot analyses of GALR3 mRNA in brain and variousperipheral rat tissues. Intensity Therapeutic Tissue of SignalIndications Heart (−) Brain (−) Spleen (−) Lung (−) Liver + DiabetesSkeletal Muscle (−) Kidney ++ Hypertension, electrolyte balance,diuretic, anti-diuretic Testis (−) Spinal cord (−) Periaqueductal Grey(−) Cerebellum (−) Cortex (−) Brain Stem (−) Hypothalamus (−) Amygdala(−) RIN14B cell line (−)

TABLE 2 RT-PCR analyses of GALR3 mRNA in brain and various peripheralrat tissues. Intensity Tissue of Signal Therapeutic Indications Heart(−) Brain + Obesity/feeding, analgesia, cognition enhancement,Alzheimer's disease, depression, anxiety, sleep disorders, Parkinson'sdisease, traumatic brain injury, convulsion/epilepsy Spleen + Immunefunctions, hematopoiesis Lung + Respiratory disorders, asthma,emphysema, lung cancer diagnostics Liver + Diabetes Skeletal Muscle (−)Smooth Muscle + Diabetes Kidney + Hypertension, electrolyte balance,diuretic, anti- diuretic Pancreas +++ Appetite/obesity, diabetes,gastrointestinal disorders, neuroendocrine regulation Retina (−)Testis + Reproductive function Ventral spinal ++ Analgesia cord Dorsalspinal ++ Analgesia cord Periaqueductal (−) Grey Cerebellum + Motordisorders Cortex (−) Brain Stem + Autonomic disorders Lower midbrain +Hypothalamus ++ Neuroendocrine regulation, appetite/obesity Amygdala (−)RIN14B cell + Neuroendocrine line regulation, including diabetes

Pharmacological characterization of GALR3

The pharmacology of GALR3 was studied in COS-7 cells transientlytransfected with the GALR3 cDNA, K163-30-17 (or “K1086”). COS-7 cellstransfected with the single clone K1086 exhibit specific binding of¹²⁵I-galanin in comparison with COS-7 cells transfected with controlvector. In preliminary radioligand binding experiments, porcine¹²⁵I-galanin bound to membranes from COS-7 cells transfected with K1086,with a specific binding of 90 fmol/mg, when the membranes (0.17 mg/mL)were incubated with 2.1 nM porcine 125I-galanin for 60 min at roomtemperature. (Specific binding was decreased by as much as 70% when theincubation temperature was raised to 30° C., suggesting receptorinstability and/or protease activity in the membrane preparation.) Inthis experiment, the binding buffer used was that described for thewhole cell slide binding assay. No specific binding was detected tomembranes from mock-transfected COS-7 cells when tested under the sameconditions.

In another experiment, COS-7 cells were transiently transfected with a“trimmed” plasmid (designated pEXJ-RGalR3T), which comprises the entirecoding region of rat GALR3, but in which the 5′ initiating ATG is joineddirectly to the vector, and which comprises only 100 nucleotides fromthe 3′ untranslated region, after the stop codon (i.e., up to andincluding nucleotide 1275 in FIG. 1). A full saturation binding analysisusing 125I-galanin was performed using the COS-7 cells transfected withplasmid pEXJ-RGalR3T, and yielded a K_(d) (dissociation constant) of0.34 nM and an apparent B_(max) as high as 570 fmol/mg. The use of the“trimmed” plasmid provides for greater expression and therefore greaterconvenience and accuracy in binding assays.

Peptide displacement assays yielded a distinct rank order of bindingaffinity (Table 3) Porcine galanin bound with relatively high affinity(K_(i)=5 nM), C-terminal truncation to porcine galanin 1-16 wasdisruptive (Ki=86 nM), and galanin 3-29 as well as D-Trp²-galaninanalogs were without demonstrable binding. Two chimeric peptidesdisplayed high affinity for GALR3 (M32 and M35) whereas galantide wasslightly less active and the putative “antagonists” C7 and M40 wererelatively weak ligands.

Peptide binding profiles for the rat GALR1, GALR2 and GALR3 receptorsubtypes were derived from membranes prepared from transientlytransfected COS-7 cells. Rat GALR3 is distinguished from the otherreceptor subtypes by having 40-fold lower affinity for M40 vs. galanin,whereas the rat GALR1 and GALR2 receptor subtypes display <=8-fold loweraffinity for M40 vs. galanin. Rat GALR3 also displays low affinity forthe D-Trp²-galanin analogs, which appear to be primarily useful fordistinguishing the rat GALR2 receptor. It is concluded that the ratGALR3 displays a distinctive pharmacological profile which can be usedto evaluate receptor expression in native cells and tissues.

Table 3. Peptide binding profile of rat GALR1, GALR2 and GALR3 receptorstransiently expressed in COS-7 cell membranes and labeled with porcine¹²⁵I-galanin. Values are reported as K_(i) (nM).

GALR1 (K_(i), GALR2 (K_(i), GALR3 (K_(i), Peptide nM) nM) nM) porcine0.46 0.45 5.1 galanin M32 0.62 12 2.1 M35 0.33 0.57 6.7 galantide 9.52.0 18 C7 16 19 68 M40 3.6 0.72 210 porcine 2.2 7.2 86 galanin 1-16D-Trp²-galanin 3700 52 >1000 1-29 D-Trp²-galanin 40 000 23 >1000 1-16porcine >100 000 >100 000 >1000 galanin 3-29

Isolation of the Human GALR3 Gene

A human placenta genomic library in λ dash II (≈1.5×10⁶ totalrecombinants) was screened using the same set of overlappingoligonucleotide probes to TM regions 1-7 of rat GALR2 and under the samehybridization and wash conditions as described for screening the rathypothalamus cDNA library. Lambda phage clones hybridizing with theprobe were plaque purified and DNA was prepared for Southern blotanalysis. One phage clone, plc21a, contained a 2.7 kb KpnI/EcoRIfragment which hybridized with the rat GALR2 TM2 oligonucleotide probeand was subsequently subcloned into a pUC vector for sequence analysis.The cloned human genomic fragment contains an a open reading frame fromthe starting MET codon to a predicted intron in the second intracellularloop, with a nucleotide identity of 88% (93% aa identity) with the ratGALR3 receptor described above (thus establishing this human genomicclone to be the human homologue of rat GALR3). Although this humangenomic fragment was not full-length and contained an intron downstreamof TM3, it is anticipated that the full-length, intronless version ofthe human GALR3 receptor gene may be isolated using standard molecularbiology techniques, as described in Materials and Methods.

Since the human genomic fragment was not full-length and contained anintron downstream of TM3, it was hypothesized that the original phageclone, which contains an average insert size of about 18 kb, may containthe 3′ end of this gene, assuming a smaller size for the intron whichserparates the 5′ and 3′ exons. The presence of the exon, representingthe 3′ end of the human GALR3, on the original phage clone, wasdemonstrated by positive hybridization signals of the phage clone,plc21a, with probes directed to the third extracellular loop or TM4 ofthe rat GALR3 gene.

The full-length human GALR3 gene was constructed by ligating aPCR-derived product of the 5′ exon, representing the starting METthrough the 3/4 loop with a synthetically-created KpnI site appended tothe reverse PCR primer, and the 3′ exon, contained on a 1.4 kb KpnIgenomic fragment. The full-length human GALR3 gene contains 1107 bpwithin its coding region, encoding for a predicted protein of 368 aa.The rat homologue contains two additional aa and encodes for a predictedprotein of 370 aa. The human and rat GALR3 homologues exhibit 86%nucleotide and 92% amino acid identities, consistent with designatingthese genes as species homologues of the same gene within the GPCRfamily. The amino acid identity increases to 96% when restricting thecomparison to within the transmembrane domains. The human GALR3 geneexhibits 52% and 67% nucleotide identities and 36% and 58% amino acididentities to the human GALR1 and GALR2 receptors, respectively.Furthermore, within the transmembrane domains, the human GALR3 receptordisplays 46% and 74% amino acid identities with the human GALR1 andGALR2 receptors, respectively. This relationship suggests that humanGALR3 represents a novel receptor subtype within the galanin genefamily.

Pharmacological Characterization of Human GALR3

The pharmacology of human GALR3 was studied in COS-7 cells transientlytransfected with pEXJ-hGalR3. In preliminary radioligand bindingexperiments using membranes prepared from COS-7 cells transfected withpEXJ-hGalR3, specific binding of galanin was observed with binding of 6fmol/mg when the membranes (0.31 mg/mL) were incubated with 0.32 nMporcine ¹²⁵I-galanin for 2 hrs. at room temperature. No mocktransfection was performed in this assay because no galanin binding toCOS-7 cells was observed previously in binding experiments using similarconditions (supra).

In a subsequent experiment, when membranes from transiently transfectedcells (membrane protein =0.15 mg/ml) were incubated with porcine¹²⁵I-galanin (0.32 nM), specific binding was measured as 110 fmol/mg.Therefore, it is concluded that the human GALR3 receptor cDNA leads toexpression of functional GALR3 receptors, thereby providing an importanttool with which to evaluate ligand selectivity for human GALR1, GALR2and GALR3 receptor subtypes.

In further experiments, cell lines stably expressing the rat and humanGALR3 receptors were prepared. Membranes from the stably transfectedcell line 293-rGalR3-105 bound porcine ¹²⁵I-galanin with a K_(d) of 0.74nM and an apparent B_(max) of 450 fmol/mg membrane protein. Both thetransiently and stably expressed rat GALR3 receptors were analyzed incompetitive displacement assays using porcine ¹²⁵I-galanin (Table 4).Like GALR2, GALR3 appears to bind the N-terminally extended peptidegalanin −7 to +29 with affinity comparable to that for porcine galanin.These data provide a pharmacological fingerprint which should be usefulfor characterizing GALR3-dependent processes in vivo.

Next, the cDNA for the human GALR3 receptor was used to prepare bothtransiently and stably transfected cells. Membranes from COS-7 cellstransiently transfected with human GALR3 cDNA bound porcine ¹²⁵I-galaninwith a K_(d) of 1.25 nM and an apparent B_(max) of 750 fmol/mg membraneprotein. Membranes LM(tk-) cells stably transfected with human GALR3receptor cDNA (L-hGalR3-228) bound porcine ¹²⁵I-galanin with a K_(d) of2.57 nM and an apparent B_(max) of 1700 fmol/mg membrane protein.Preliminary analyses in peptide displacement assays using porcine¹²⁵I-galanin as the radioligand indicate that the human GALR3 receptor,sharing 92% amino acid identity with the rat GALR3 receptor, bindsgalanin and related analogs with affinities resembling those for the ratreceptor. A similar pharmacological profile for both the human and ratGALR3 receptor homologs suggests that the rat may be used to model thetherapeutic value of GALR3-directed ligands.

TABLE 4 Ki (nM) Rat Human GalR3 293-rGalR3- GalR3 L-hGalR3- Peptide COS7105 COS7 228 M32 1.9 1.0 M35 3.7 3.2 7.8 rat 4.3 5.7 galanin porcine 5.15.8 5.3 14 galanin galantide 9.0 7.6 C-7 23 9.6 M40 103 85 130 porcine52 138 35 galanin 1- 16 D-Trp2- >1000 >1000 galanin galanin 3.3 −7 to+29

Signal Transduction Pathway of hGalR3: Stimulation of K⁺ Currents

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987; Dascal etal., 1993). A large family of GPCRs that naturally couple toheterotrimeric G-proteins of the G_(i)/G_(o) class activate GIRKchannels (North, 1989) in native neurons (Kofuji et al., 1995) and inthe Xenopus expression system (Dascal et al., 1993; Kubo et al., 1993;Krapavinsky et al., 1995). Under voltage clamp conditions, oocytesinjected with mRNAs for hGALR3 and GIRKs 1 and 4 responded with inwardcurrents to local perfusion of porcine galanin (FIG. 6A). Averagecurrents were 51.3±9.4 nA (n=16) in the presence of 1 μM porcinegalanin, whereas oocytes injected with mRNAs for GIRKs 1 and 4 aloneproduced little or no inward current (2.5±1.2 nA, n=8) in response to 1μM galanin. Evidence that galanin-induced currents were mediated by GIRKchannels included: 1) dependency on elevated external K⁺, 2) stronginward rectification of the current-voltage (I/V) relation, 3) reversalpotential close to the predicted equilibrium potential for K⁺, 4)sensitivity to block by 300 μM Ba⁺⁺ (FIG. 6A), and 5) lack ofgalanin-sensitivity in oocytes injected with only hGALR3 mRNA (data notshown). Currents having these same properties, but larger in amplitude,were also evoked by galanin in oocytes expressing GALR1 receptors incombination with GIRKs 1 and 4 (data not shown). Thus, GALR1 and GALR3receptors appear to have a related signal transduction pathway.

Other GPCRs, when expressed in Xenopus oocytes, activate aCa⁺⁺-dependent Cl⁻ conductance that results from the activation ofphospholipase C and the subsequent release of Ca⁺⁺ from intracellularstores. This pathway was not activated in oocytes expressing hGALR3since Cl⁻ currents were never observed following application of galanin(n=20). (Cl⁻ currents were also not observed in oocytes expressing theGALR1 receptor.) In contrast, in oocytes expressing mRNAs encoding GALR2or Ala receptors, 1 μM galanin or epinephrine, respectively, stimulatestransient Cl⁻ currents (data not shown). To provide further evidencethat hGALR3 couples to the G_(o)/G_(i) family of G-proteins, batches ofoocytes, previously injected with hGALR3 and GIRK mRNAs, were injectedwith pertussis toxin (2 ng/oocyte) and tested for receptor coupling toK⁺ currents. In oocytes treated with the toxin, galanin currents werecompletely abolished (FIG. 7); oocytes injected with buffer alonedisplayed normal galanin-induced currents. A similar sensitivity topertussis toxin was observed for oocytes expressing GALR1 receptors.Agonist responses in oocytes expressing GALR2 or α_(1a) adrenergicreceptors were unaffected by pertussis toxin (FIG. 7). Taken together,these results support the conclusion that GALR1 and GALR3 receptorscouple to a Gi/Go pathway, and that GALR2 (like the α_(1a) adrenergicreceptor) couples to a G_(q)-type pathway (Table 5).

TABLE 5 Comparison of intracellular signaling pathways for three galaninreceptors expressed in oocytes. Signaling pathway Activates ActivatesReceptor Cl⁻ current GIRKs PTX sensitive rGALR1 no yes yes rGALR2 yes nono hGALR3 no yes yes

Pharmacology of hGALR3 in Oocytes

A series of galanin and galanin-related peptides were tested at thehuman GALR3 receptor for agonist and antagonist activities. Of thesepeptides, porcine galanin, human galanin, M32, C7, M35, M15 (spantide),galanin −7-29, galanin 1-16, and M40 evoked agonist activity at a fixeddose of 1 μM. D-Trp2-galanin and galanin 3-29 were inactive. EC₅₀s wereconstructed from cumulative concentration-response measurementsperformed on a series of oocytes (FIGS. 6B, 8). EC₅₀s (in rank order)for M32, porcine galanin, C7, galanin −7-29, galanin 1-16 and M40 were44.5, 222, 343, 1906, 2030, and 2265 nM, respectively (Table 6). Thisrank order of potency was similar to that observed for K_(i) values inbinding assays using the rat GalR3 receptor in COS-7 cells with theexception of galanin −7-29, which had an affinity between galanin andM32 in the binding assay.

We have observed that the peptide galanin −7-29, which binds selectivelyto GALR3 over GALR1 and to GALR2 over GALR1, induces feeding in ratswhen injected i.c.v. Another peptide, shown in binding and functionalstudies to selectively bind to the GALR2 receptor over both GALR1 andGALR3, did not stimulate feeding. Taken together, these results suggesta role for GALR3 in mediating galanin-induced feeding.

TABLE 6 Comparison of rank orders of EC₅₀S for stimulation of GIRKs,apparent binding affinities (K_(i)) and EC₅₀s for stimulation of feedingbehavior in vivo. Cos-7 K_(i) Oocyte EC₅₀ (rat GalR3) Peptide (nM) (nM)M32 45 1.9 p-Galanin 222 5.1 C7 343 23.0 gal −7 to 29 1,906 3.3 gal 1-162,030 51.9 M40 2,265 103.0

Experimental Discussion

Using a combination of homology and expression cloning strategies,nucleic acids have been isolated encoding a novel galanin receptor,termed GALR3, that is distinct from the previously cloned GALR1 andGALR2 receptors.

The rat GALR3 gene, whose sequence is derived from cDNA, does not haveany other MET upstream of the proposed starting MET, in any of the threeopen reading frames.

The human GALR3 gene contains two in-frame METS: the first (as one reads5′ to 3′) will be referred to herein as the “upstream MET” and thesecond (i.e., closer to TM1) will be referred to herein as the“downstream MET.” Both the upstream and downstream METs are shown inFIG. 4 (Seq. ID No. 4). Based on data currently available, it isbelieved that the downstream MET is likely to be the correct initiatingmethionine. It is theoretically possible that the upstream MET might bethe initiating MET. It is to be understood that the present inventionincludes both the receptor beginning at the downstream MET and thereceptor beginning at the upstream MET.

The existence of multiple galanin receptor subtypes suggests thepotential for the design and discovery of novel subtype selectivecompounds. In this regard, the expression of the cDNA encoding the GALR3receptor in cultured cell lines and other cells provides a unique toolfor the discovery of therapeutic agents targeted at galanin receptors.

The localization of GALR1 receptors to multiple brain regions(Gustafson, et al., 1996; Parker, et al., 1995) and the identificationof GALR3 in a hypothalamic cDNA library, suggests multiple therapeuticindications for the use of galanin receptor-selective drugs. Theseinclude feeding, cognition, analgesia and/or sensory processing, andanxiety and depression.

The observation that galanin is co-released with norepinephrine fromsympathetic nerve terminals suggests that galanin could act via galaninreceptors in the periphery to modulate nearly every physiologicalprocess controlled by sympathetic innervation. Additional therapeuticindications not directly related to localization include diabetes,hypertension, cardiovascular disorders, regulation of growth hormonerelease, regulation of fertility, gastric ulcers, gastrointestinalmotility/transit/absorption/secretion, glaucoma, inflammation, immunedisorders, respiratory disorders (e.g., asthma, emphysema).

The physiological and anatomical distribution of galanin-containingneurons suggests potential roles of galanin receptors mediating effectson cognition, analgesia, neuroendocrine regulation, control of insulinrelease and control of feeding behavior. Of particular relevance to therole of the novel GALR3 receptor, are those functions mediated bygalanin receptors in the rat hypothalamus.

Studies in rats indicate that the injection of galanin in thehypothalamus increases food intake (Kyrouli et al, 1990, and Schick etal, 1993) and that this stimulatory effect of galanin is blocked byprior administration of M40 and C7 (Liebowitz and Kim, 1992; and Corwin,1993). The expression of the mRNA encoding the GALR1 receptor in the rathypothalamus (Parker et al., 1995; Gustafson et al., 1996), and the factthat the novel GALR3 receptor was identified in a cDNA library preparedfrom rat hypothalamus argues in favor of the involvement of one or moregalanin receptor subtypes in the regulation of feeding behavior.However, the evidence against the involvement of GALR1 in thestimulation of feeding behavior stems from the fact that M40 and C7 areknown to be agonists, and not antagonists, in cell lines expressinghuman and rat GALR1 receptors (Heuillet et al. 1994; Hale et al. 1993;and Bartfai et al. 1993).

Peptide displacement assays indicate that the rat GALR3 receptor has aunique pharmacological profile. The low affinity for M40, in particular,invites further speculation as to the physiological role of the ratGALR3 receptor. It is noted that M40 was reported to be inactive, forexample, when tested for antagonism of galaninergic inhibition ofglucose-stimulated insulin release in rat pancreas, (Bartfai, 1993). Inanother example, intrathecal M40 was a weak antagonist of thegalanin-facilitated flexor reflex in rat (Xu, 1995). It was observed infeeding assays that M40 was less potent but as effective as galanin instimulating food intake when injected icv into rat brain. The data areconsistent with a role for the GALR3 receptor in a range of physiologicand pathophysiologic functions including diabetes, pain, obesity andeating disorders, and furthermore suggest that the rat GALR3 receptormay represent a target for the design of therapeutic compounds. Thecloning of the rat GALR3 receptor further enables the design anddevelopment of in vitro functional assays to determine the agonist orantagonist properties of peptides and drug development candidates.

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SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 59(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1280 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:1: AGCTCCAGCC TAGGCGTTCT ACCTGGAAGA ATGCAGGGGC CCAGTACCTA GGACTGAGGA60 AGATGGCTGA CATCCAGAAC ATTTCGCTGG ACAGCCCAGG GAGCGTAGGG GCTGTGGCAG 120TGCCTGTGAT CTTTGCCCTC ATCTTCCTGT TGGGCATGGT GGGCAATGGG CTGGTGTTGG 180CTGTGCTACT GCAGCCTGGC CCAAGTGCCT GGCAGGAGCC AAGCAGTACC ACAGATCTCT 240TCATCCTCAA CTTGGCCGTG GCCGACCTTT GCTTCATCCT GTGCTGCGTG CCCTTCCAGG 300CAGCCATCTA CACACTGGAT GCCTGGCTCT TTGGGGCTTT CGTGTGCAAG ACGGTACATC 360TGCTCATCTA CCTCACCATG TATGCCAGCA GCTTCACCCT GGCGGCCGTC TCCCTGGACA 420GGTACCTGGC TGTGCGGCAC CCACTGCGCT CCAGAGCCCT GCGCACCCCG CGCAACGCGC 480GCGCCGCCGT GGGGCTCGTG TGGCTGCTGG CGGCTCTCTT TTCCGCGCCC TACCTAAGCT 540ATTACGGCAC GGTGCGCTAC GGCGCGCTCG AGCTCTGCGT GCCCGCTTGG GAGGACGCGC 600GGCGGCGCGC GCTGGACGTG GCCACCTTCG CCGCGGGCTA CCTGCTGCCG GTGGCCGTGG 660TGAGCCTGGC CTACGGACGC ACGCTATGTT TCCTATGGGC CGCCGTGGGT CCCGCGGGCG 720CGGCGGCAGC AGAGGCGCGC AGACGGGCGA CCGGCCGGGC GGGACGCGCC ATGCTGGCAG 780TGGCCGCGCT CTACGCGCTT TGCTGGGGCC CGCACCACGC GCTCATCCTC TGCTTCTGGT 840ACGGCCGCTT CGCCTTCAGC CCGGCCACCT ACGCCTGTCG CCTGGCCTCG CACTGCCTCG 900CCTACGCCAA CTCCTGCCTT AACCCGCTCG TCTACTCGCT CGCCTCGCGC CACTTCCGCG 960CGCGCTTCCG CCGCCTGTGG CCCTGCGGCC GTCGCCGCCA CCGCCACCAC CACCGCGCTC 1020ATCGAGCCCT CCGTCGTGTC CAGCCGGCGT CTTCGGGCCC CGCCGGTTAT CCCGGCGACG 1080CCAGGCCTCG TGGTTGGAGT ATGGAGCCCA GAGGGGATGC TCTGCGTGGT GGTGGAGAGA 1140CTAGACTAAC CCTGTCCCCC AGGGGACCTC AATAACCCTG CCCGCTTGGA CTCTGACGTC 1200TGTCAGAATG CCACCAAGGA ACATCTAGGG AACGGCAGTC TCGCCAGGCT CCACCAAAAA 1260GCAGAAGCAA AGTTGCAGGG 1280 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 370 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:2: Met Ala Asp Ile Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 15 10 15 Ala Val Ala Val Pro Val Ile Phe Ala Leu Ile Phe Leu Leu Gly Met20 25 30 Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser35 40 45 Ala Trp Gln Glu Pro Ser Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu50 55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala65 70 75 80 Ala Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Phe Val CysLys 85 90 95 Thr Val His Leu Leu Ile Tyr Leu Thr Met Tyr Ala Ser Ser PheThr 100 105 110 Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Val Arg HisPro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg AlaAla Val Gly 130 135 140 Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala ProTyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr Val Arg Tyr Gly Ala Leu GluLeu Cys Val Pro Ala Trp 165 170 175 Glu Asp Ala Arg Arg Arg Ala Leu AspVal Ala Thr Phe Ala Ala Gly 180 185 190 Tyr Leu Leu Pro Val Ala Val ValSer Leu Ala Tyr Gly Arg Thr Leu 195 200 205 Cys Phe Leu Trp Ala Ala ValGly Pro Ala Gly Ala Ala Ala Ala Glu 210 215 220 Ala Arg Arg Arg Ala ThrGly Arg Ala Gly Arg Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu TyrAla Leu Cys Trp Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe TrpTyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg LeuAla Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 LeuVal Tyr Ser Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300Leu Trp Pro Cys Gly Arg Arg Arg His Arg His His His Arg Ala His 305 310315 320 Arg Ala Leu Arg Arg Val Gln Pro Ala Ser Ser Gly Pro Ala Gly Tyr325 330 335 Pro Gly Asp Ala Arg Pro Arg Gly Trp Ser Met Glu Pro Arg GlyAsp 340 345 350 Ala Leu Arg Gly Gly Gly Glu Thr Arg Leu Thr Leu Ser ProArg Gly 355 360 365 Pro Gln 370 (2) INFORMATION FOR SEQ ID NO:3: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1417 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3: Cys Ala Cys Thr Cys Ala Gly Cys Gly Ala Thr Gly Ala Cys ThrThr 1 5 10 15 Thr Gly Gly Cys Thr Cys Thr Gly Cys Thr Cys Thr Cys CysCys Cys 20 25 30 Thr Cys Cys Thr Cys Cys Ala Thr Cys Thr Cys Cys Cys AlaCys Gly 35 40 45 Ala Gly Cys Thr Thr Cys Cys Ala Gly Cys Cys Cys Ala GlyAla Ala 50 55 60 Cys Ala Cys Cys Thr Gly Gly Cys Cys Ala Gly Ala Cys CysCys Ala 65 70 75 80 Gly Gly Thr Cys Gly Gly Gly Gly Gly Ala Gly Thr ThrAla Gly Ala 85 90 95 Thr Cys Cys Cys Gly Gly Gly Gly Thr Cys Ala Ala GlyCys Ala Ala 100 105 110 Cys Cys Ala Gly Ala Ala Cys Thr Gly Gly Gly GlyGly Cys Thr Cys 115 120 125 Thr Thr Gly Cys Cys Thr Gly Ala Gly Gly AlaThr Thr Cys Cys Ala 130 135 140 Gly Cys Thr Thr Cys Thr Cys Thr Thr CysCys Cys Ala Gly Gly Thr 145 150 155 160 Gly Cys Cys Cys Gly Thr Cys ThrGly Ala Thr Gly Gly Gly Gly Ala 165 170 175 Gly Ala Thr Gly Gly Cys ThrGly Ala Thr Gly Cys Cys Cys Ala Gly 180 185 190 Ala Ala Cys Ala Thr ThrThr Cys Ala Cys Thr Gly Gly Ala Cys Ala 195 200 205 Gly Cys Cys Cys AlaGly Gly Gly Ala Gly Thr Gly Thr Gly Gly Gly 210 215 220 Gly Gly Cys CysGly Thr Gly Gly Cys Ala Gly Thr Gly Cys Cys Thr 225 230 235 240 Gly ThrGly Gly Thr Cys Thr Thr Thr Gly Cys Cys Cys Thr Ala Ala 245 250 255 ThrCys Thr Thr Cys Cys Thr Gly Cys Thr Gly Gly Gly Cys Ala Cys 260 265 270Ala Gly Thr Gly Gly Gly Cys Ala Ala Thr Gly Gly Gly Cys Thr Gly 275 280285 Gly Thr Gly Cys Thr Gly Gly Cys Ala Gly Thr Gly Cys Thr Cys Cys 290295 300 Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Cys Cys Cys Gly Ala Gly305 310 315 320 Thr Gly Cys Cys Thr Gly Gly Cys Ala Gly Gly Ala Gly CysCys Thr 325 330 335 Gly Gly Cys Ala Gly Cys Ala Cys Cys Ala Cys Gly GlyAla Cys Cys 340 345 350 Thr Gly Thr Thr Cys Ala Thr Cys Cys Thr Cys AlaAla Cys Cys Thr 355 360 365 Gly Gly Cys Gly Gly Thr Gly Gly Cys Thr GlyAla Cys Cys Thr Cys 370 375 380 Thr Gly Cys Thr Thr Cys Ala Thr Cys CysThr Gly Thr Gly Cys Thr 385 390 395 400 Gly Cys Gly Thr Gly Cys Cys CysThr Thr Cys Cys Ala Gly Gly Cys 405 410 415 Cys Ala Cys Cys Ala Thr CysThr Ala Cys Ala Cys Gly Cys Thr Gly 420 425 430 Gly Ala Thr Gly Cys CysThr Gly Gly Cys Thr Cys Thr Thr Thr Gly 435 440 445 Gly Gly Gly Cys CysCys Thr Cys Gly Thr Cys Thr Gly Cys Ala Ala 450 455 460 Gly Gly Cys CysGly Thr Gly Cys Ala Cys Cys Thr Gly Cys Thr Cys 465 470 475 480 Ala ThrCys Thr Ala Cys Cys Thr Cys Ala Cys Cys Ala Thr Gly Thr 485 490 495 AlaCys Gly Cys Cys Ala Gly Cys Ala Gly Cys Thr Thr Thr Ala Cys 500 505 510Gly Cys Thr Gly Gly Cys Thr Gly Cys Thr Gly Thr Cys Thr Cys Cys 515 520525 Gly Thr Gly Gly Ala Cys Ala Gly Gly Thr Ala Cys Cys Thr Gly Gly 530535 540 Cys Cys Gly Thr Gly Cys Gly Gly Cys Ala Cys Cys Cys Gly Cys Thr545 550 555 560 Gly Cys Gly Cys Thr Cys Gly Cys Gly Cys Gly Cys Cys CysThr Gly 565 570 575 Cys Gly Cys Ala Cys Gly Cys Cys Gly Cys Gly Thr AlaAla Cys Gly 580 585 590 Cys Cys Cys Gly Cys Gly Cys Cys Gly Cys Ala GlyThr Gly Gly Gly 595 600 605 Gly Cys Thr Gly Gly Thr Gly Thr Gly Gly CysThr Gly Cys Thr Gly 610 615 620 Gly Cys Gly Gly Cys Gly Cys Thr Cys ThrThr Cys Thr Cys Gly Gly 625 630 635 640 Cys Gly Cys Cys Cys Thr Ala CysCys Thr Cys Ala Gly Cys Thr Ala 645 650 655 Cys Thr Ala Cys Gly Gly CysAla Cys Cys Gly Thr Gly Cys Gly Cys 660 665 670 Thr Ala Cys Gly Gly CysGly Cys Gly Cys Thr Gly Gly Ala Gly Cys 675 680 685 Thr Cys Thr Gly CysGly Thr Gly Cys Cys Cys Gly Cys Cys Thr Gly 690 695 700 Gly Gly Ala GlyGly Ala Cys Gly Cys Gly Cys Gly Cys Cys Gly Cys 705 710 715 720 Cys GlyCys Gly Cys Cys Cys Thr Gly Gly Ala Cys Gly Thr Gly Gly 725 730 735 CysCys Ala Cys Cys Thr Thr Cys Gly Cys Thr Gly Cys Cys Gly Gly 740 745 750Cys Thr Ala Cys Cys Thr Gly Cys Thr Gly Cys Cys Cys Gly Thr Gly 755 760765 Gly Cys Thr Gly Thr Gly Gly Thr Gly Ala Gly Cys Cys Thr Gly Gly 770775 780 Cys Cys Thr Ala Cys Gly Gly Gly Cys Gly Cys Ala Cys Gly Cys Thr785 790 795 800 Gly Cys Gly Cys Thr Thr Cys Cys Thr Gly Thr Gly Gly GlyCys Cys 805 810 815 Gly Cys Cys Gly Thr Gly Gly Gly Thr Cys Cys Cys GlyCys Gly Gly 820 825 830 Gly Cys Gly Cys Gly Gly Cys Gly Gly Cys Gly GlyCys Cys Gly Ala 835 840 845 Gly Gly Cys Gly Cys Gly Gly Cys Gly Gly AlaGly Gly Gly Cys Gly 850 855 860 Ala Cys Gly Gly Gly Cys Cys Gly Cys GlyCys Gly Gly Gly Gly Cys 865 870 875 880 Gly Cys Gly Cys Cys Ala Thr GlyCys Thr Gly Gly Cys Gly Gly Thr 885 890 895 Gly Gly Cys Cys Gly Cys GlyCys Thr Cys Thr Ala Cys Gly Cys Gly 900 905 910 Cys Thr Cys Thr Gly CysThr Gly Gly Gly Gly Thr Cys Cys Gly Cys 915 920 925 Ala Cys Cys Ala CysGly Cys Gly Cys Thr Cys Ala Thr Cys Cys Thr 930 935 940 Gly Thr Gly CysThr Thr Cys Thr Gly Gly Thr Ala Cys Gly Gly Cys 945 950 955 960 Cys GlyCys Thr Thr Cys Gly Cys Cys Thr Thr Cys Ala Gly Cys Cys 965 970 975 CysGly Gly Cys Cys Ala Cys Cys Thr Ala Cys Gly Cys Cys Thr Gly 980 985 990Cys Cys Gly Cys Cys Thr Gly Gly Cys Cys Thr Cys Ala Cys Ala Cys 995 10001005 Thr Gly Cys Cys Thr Gly Gly Cys Cys Thr Ala Cys Gly Cys Cys Ala1010 1015 1020 Ala Cys Thr Cys Cys Thr Gly Cys Cys Thr Cys Ala Ala CysCys Cys 1025 1030 1035 1040 Gly Cys Thr Cys Gly Thr Cys Thr Ala Cys GlyCys Gly Cys Thr Cys 1045 1050 1055 Gly Cys Cys Thr Cys Gly Cys Gly CysCys Ala Cys Thr Thr Cys Cys 1060 1065 1070 Gly Cys Gly Cys Gly Cys GlyCys Thr Thr Cys Cys Gly Cys Cys Gly 1075 1080 1085 Cys Cys Thr Gly ThrGly Gly Cys Cys Gly Thr Gly Cys Gly Gly Cys 1090 1095 1100 Cys Gly CysCys Gly Ala Cys Gly Cys Cys Gly Cys Cys Ala Cys Cys 1105 1110 1115 1120Gly Thr Gly Cys Cys Cys Gly Cys Cys Gly Cys Gly Cys Cys Thr Thr 11251130 1135 Gly Cys Gly Thr Cys Gly Cys Gly Thr Cys Cys Gly Cys Cys CysCys 1140 1145 1150 Gly Cys Gly Thr Cys Cys Thr Cys Gly Gly Gly Cys CysCys Ala Cys 1155 1160 1165 Cys Cys Gly Gly Cys Thr Gly Cys Cys Cys CysGly Gly Ala Gly Ala 1170 1175 1180 Cys Gly Cys Cys Cys Gly Gly Cys CysThr Ala Gly Cys Gly Gly Gly 1185 1190 1195 1200 Ala Gly Gly Cys Thr GlyCys Thr Gly Gly Cys Thr Gly Gly Thr Gly 1205 1210 1215 Gly Cys Gly GlyCys Cys Ala Gly Gly Gly Cys Cys Cys Gly Gly Ala 1220 1225 1230 Gly CysCys Cys Ala Gly Gly Gly Ala Gly Gly Gly Ala Cys Cys Cys 1235 1240 1245Gly Thr Cys Cys Ala Cys Gly Gly Cys Gly Gly Ala Gly Ala Gly Gly 12501255 1260 Cys Thr Gly Cys Cys Cys Gly Ala Gly Gly Ala Cys Cys Gly GlyAla 1265 1270 1275 1280 Ala Thr Ala Ala Ala Cys Cys Cys Thr Gly Cys CysGly Cys Cys Thr 1285 1290 1295 Gly Gly Ala Cys Thr Cys Cys Gly Cys CysThr Gly Thr Gly Thr Cys 1300 1305 1310 Cys Gly Thr Cys Thr Gly Thr CysThr Cys Ala Cys Thr Cys Cys Cys 1315 1320 1325 Gly Thr Thr Cys Thr CysCys Gly Ala Ala Gly Gly Cys Gly Gly Gly 1330 1335 1340 Ala Cys Gly CysCys Ala Cys Cys Gly Gly Gly Cys Cys Ala Gly Gly 1345 1350 1355 1360 GlyAla Thr Gly Gly Gly Gly Cys Ala Ala Thr Gly Cys Cys Ala Cys 1365 13701375 Gly Ala Gly Cys Thr Cys Thr Cys Thr Gly Ala Gly Gly Gly Gly Cys1380 1385 1390 Gly Thr Thr Gly Ala Gly Thr Gly Gly Ala Gly Cys Gly AlaCys Thr 1395 1400 1405 Thr Gly Thr Cys Cys Cys Cys Gly Cys 1410 1415 (2)INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:427 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: His Ser Ala MetThr Leu Ala Leu Leu Ser Pro Pro Pro Ser Pro Thr 1 5 10 15 Ser Phe GlnPro Arg Thr Pro Gly Gln Thr Gln Val Gly Gly Val Arg 20 25 30 Ser Arg GlyGln Ala Thr Arg Thr Gly Gly Ser Cys Leu Arg Ile Pro 35 40 45 Ala Ser LeuPro Arg Cys Pro Ser Asp Gly Glu Met Ala Asp Ala Gln 50 55 60 Asn Ile SerLeu Asp Ser Pro Gly Ser Val Gly Ala Val Ala Val Pro 65 70 75 80 Val ValPhe Ala Leu Ile Phe Leu Leu Gly Thr Val Gly Asn Gly Leu 85 90 95 Val LeuAla Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu Pro 100 105 110 GlySer Thr Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp Leu 115 120 125Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr Leu 130 135140 Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys Ala Val His Leu Leu 145150 155 160 Ile Tyr Leu Thr Met Tyr Ala Ser Ser Phe Thr Leu Ala Ala ValSer 165 170 175 Val Asp Arg Tyr Leu Ala Val Arg His Pro Leu Arg Ser ArgAla Leu 180 185 190 Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly Leu ValTrp Leu Leu 195 200 205 Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr TyrGly Thr Val Arg 210 215 220 Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala TrpGlu Asp Ala Arg Arg 225 230 235 240 Arg Ala Leu Asp Val Ala Thr Phe AlaAla Gly Tyr Leu Leu Pro Val 245 250 255 Ala Val Val Ser Leu Ala Tyr GlyArg Thr Leu Arg Phe Leu Trp Ala 260 265 270 Ala Val Gly Pro Ala Gly AlaAla Ala Ala Glu Ala Arg Arg Arg Ala 275 280 285 Thr Gly Arg Ala Gly ArgAla Met Leu Ala Val Ala Ala Leu Tyr Ala 290 295 300 Leu Cys Trp Gly ProHis His Ala Leu Ile Leu Cys Phe Trp Tyr Gly 305 310 315 320 Arg Phe AlaPhe Ser Pro Ala Thr Tyr Ala Cys Arg Leu Ala Ser His 325 330 335 Cys LeuAla Tyr Ala Asn Ser Cys Leu Asn Pro Leu Val Tyr Ala Leu 340 345 350 AlaSer Arg His Phe Arg Ala Arg Phe Arg Arg Leu Trp Pro Cys Gly 355 360 365Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu Arg Arg Val Arg Pro 370 375380 Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp Ala Arg Pro Ser Gly 385390 395 400 Arg Leu Leu Ala Gly Gly Gly Gln Gly Pro Glu Pro Arg Glu GlyPro 405 410 415 Val His Gly Gly Glu Ala Ala Arg Gly Pro Glu 420 425 (2)INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:395 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Glu Leu AlaPro Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro ProAla Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe Ile ThrLeu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45 Gly Asn SerLeu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Ala 50 55 60 Trp Gln LysPro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser 65 70 75 80 Ile AlaAsp Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr 85 90 95 Val TyrAla Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe 100 105 110 IleHis Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu 115 120 125Ala Ala Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg 130 135140 Ser Ser Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe 145150 155 160 Ile Trp Ala Leu Ser Ile Ala Met Ala Ser Pro Tyr Val Ala TyrTyr 165 170 175 Gln Arg Leu Phe His Arg Asp Ser Asn Gln Thr Phe Cys TrpGlu His 180 185 190 Trp Pro Asn Gln Leu His Lys Lys Ala Tyr Val Val CysThr Phe Val 195 200 205 Phe Gly Tyr Leu Leu Pro Leu Leu Leu Ile Cys PheCys Tyr Ala Lys 210 215 220 Val Leu Asn His Leu His Lys Lys Leu Lys AsnMet Ser Lys Lys Ser 225 230 235 240 Glu Ala Ser Lys Lys Arg Ala Thr GlyLys Thr Ala Gln Thr Val Leu 245 250 255 Val Val Val Val Val Phe Gly IleSer Trp Leu Pro His His Val Ile 260 265 270 His Leu Trp Ala Glu Phe GlyAla Phe Pro Leu Thr Pro Ala Ser Phe 275 280 285 Phe Phe Arg Ile Thr AlaHis Cys Leu Ala Tyr Ser Asn Ser Ser Val 290 295 300 Asn Pro Ile Ile TyrAla Phe Leu Ser Glu Asn Phe Arg Lys Ala Tyr 305 310 315 320 Lys Gln ValPhe Lys Cys Arg Val Cys Asn Glu Ser Pro His Gly Asp 325 330 335 Ala LysGlu Lys Asn Arg Ile Asp Thr Pro Pro Ser Thr Asn Cys Thr 340 345 350 HisVal Pro Gly Asp Ala Arg Pro Arg Gly Trp Ser Met Ala Gly Gly 355 360 365Gly Gln Gly Pro Glu Pro Arg Gly Asp Ala Leu Arg Gly Gly Gly Glu 370 375380 Thr Arg Leu Thr Leu Ser Pro Arg Gly Pro Gln 385 390 395 (2)INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Thr Thr Gly ThrAla Cys Cys Cys Cys Thr Ala Thr Thr Thr Thr Thr 1 5 10 15 Cys Gly CysGly Cys Thr Cys Ala Thr Cys Thr Thr Cys Cys Thr Cys 20 25 30 Gly Thr GlyGly Gly Cys Ala Cys Cys Gly Thr Gly Gly 35 40 45 (2) INFORMATION FOR SEQID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:7: Ala Gly Cys Ala Cys Cys Gly Cys Cys Ala GlyCys Ala Cys Cys Ala 1 5 10 15 Gly Cys Gly Cys Gly Thr Thr Gly Cys CysCys Ala Cys Gly Gly Thr 20 25 30 Gly Cys Cys Cys Ala Cys Gly Ala Gly GlyAla Ala Gly 35 40 45 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:8: Thr Cys Ala Gly Cys Ala Cys Cys Ala Cys Cys Ala Ala Cys Cys Thr 15 10 15 Gly Thr Thr Cys Ala Thr Cys Cys Thr Cys Ala Ala Cys Cys Thr Gly20 25 30 Gly Gly Cys Gly Thr Gly Gly Cys Cys Gly Ala Cys Cys Thr Gly Thr35 40 45 Gly Thr 50 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:9: Gly Gly Cys Cys Thr Gly Gly Ala Ala Ala Gly Gly Cys Ala Cys Gly 15 10 15 Cys Ala Gly Cys Ala Cys Ala Gly Gly Ala Thr Gly Ala Ala Ala Cys20 25 30 Ala Cys Ala Gly Gly Thr Cys Gly Gly Cys Cys Ala Cys Gly Cys Cys35 40 45 Cys Ala 50 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:10: Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Gly Thr Thr Cys Ala Thr 15 10 15 Thr Thr Cys Cys Thr Cys Ala Thr Cys Thr Thr Thr Cys Thr Cys Ala20 25 30 Cys Thr Ala Thr Gly Cys Ala Cys Gly Cys Cys Ala Gly 35 40 45(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Gly Gly Ala GlyAla Cys Gly Gly Cys Gly Gly Cys Cys Ala Gly Cys 1 5 10 15 Gly Thr GlyAla Ala Gly Cys Thr Gly Cys Thr Gly Gly Cys Gly Thr 20 25 30 Gly Cys AlaThr Ala Gly Thr Gly Ala Gly Ala Ala Ala 35 40 45 (2) INFORMATION FOR SEQID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:12: Ala Ala Cys Gly Cys Gly Cys Thr Gly Gly CysCys Gly Cys Cys Ala 1 5 10 15 Thr Cys Gly Gly Gly Cys Thr Cys Ala ThrCys Thr Gly Gly Gly Gly 20 25 30 Gly Cys Thr Ala Gly Cys Ala Cys Thr GlyCys Thr Cys 35 40 45 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:13: Ala Gly Thr Ala Gly Cys Thr Cys Ala Gly Gly Thr Ala Gly Gly Gly 15 10 15 Cys Cys Cys Gly Gly Ala Gly Ala Ala Gly Ala Gly Cys Ala Gly Thr20 25 30 Gly Cys Thr Ala Gly Cys Cys Cys Cys Cys Ala Gly Ala 35 40 45(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ala Gly Cys CysAla Thr Gly Gly Ala Cys Cys Thr Cys Thr Gly Cys 1 5 10 15 Ala Cys CysThr Thr Cys Gly Thr Cys Thr Thr Thr Ala Gly Cys Thr 20 25 30 Ala Cys CysThr Gly Cys Thr Gly Cys Cys Ala Gly Thr 35 40 45 (2) INFORMATION FOR SEQID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:15: Cys Gly Cys Ala Thr Ala Gly Gly Thr Cys AlaGly Ala Cys Thr Gly 1 5 10 15 Ala Gly Gly Ala Cys Thr Ala Gly Cys AlaCys Thr Gly Gly Cys Ala 20 25 30 Gly Cys Ala Gly Gly Thr Ala Gly Cys ThrAla Ala Ala 35 40 45 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:16: Gly Ala Thr Cys Ala Thr Cys Ala Thr Cys Gly Thr Gly Gly Cys Gly 15 10 15 Gly Thr Gly Cys Thr Thr Thr Thr Cys Thr Gly Cys Cys Thr Cys Thr20 25 30 Gly Thr Thr Gly Gly Ala Thr Gly Cys Cys Cys Cys Ala 35 40 45(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Cys Cys Ala CysAla Cys Gly Cys Ala Gly Ala Gly Gly Ala Thr Ala 1 5 10 15 Ala Gly CysGly Cys Gly Thr Gly Gly Thr Gly Gly Gly Gly Cys Ala 20 25 30 Thr Cys CysAla Ala Cys Ala Gly Ala Gly Gly Cys Ala 35 40 45 (2) INFORMATION FOR SEQID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:18: Gly Thr Thr Gly Cys Gly Cys Ala Thr Cys CysThr Thr Thr Cys Ala 1 5 10 15 Cys Ala Cys Cys Thr Ala Gly Thr Thr ThrCys Cys Thr Ala Thr Gly 20 25 30 Cys Cys Ala Ala Cys Thr Cys Cys Thr GlyThr Gly Thr 35 40 45 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 46 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:19: Ala Gly Ala Cys Cys Ala Gly Ala Gly Cys Gly Thr Ala Ala Ala Cys 15 10 15 Gly Ala Thr Gly Gly Gly Gly Thr Thr Gly Ala Cys Ala Cys Ala Gly20 25 30 Gly Ala Gly Thr Thr Gly Gly Cys Ala Thr Ala Gly Gly Ala 35 4045 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Cys Cys Thr CysAla Gly Thr Gly Ala Ala Gly Gly Gly Ala Ala Thr 1 5 10 15 Gly Gly GlyAla Gly Cys Gly Ala 20 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:21: Gly Thr Ala Gly Thr Gly Thr Ala Thr Ala Ala Ala Cys Thr Thr Gly 15 10 15 Cys Ala Gly Ala Thr Gly Ala Ala Gly Gly Cys 20 25 (2)INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Ala Thr Gly AlaAla Thr Gly Gly Cys Thr Cys Cys Gly Gly Cys Ala 1 5 10 15 Gly Cys CysAla Gly Gly Gly 20 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:23: Thr Thr Gly Cys Ala Gly Ala Gly Cys Ala Gly Cys Gly Ala Gly Cys 15 10 15 Cys Gly Ala Ala Cys Ala Cys 20 (2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:24: Gly Gly Cys Thr Gly Ala Cys Ala Thr Cys Cys Ala Gly AlaAla Cys 1 5 10 15 Ala Thr Thr Thr Cys Gly Cys Thr 20 (2) INFORMATION FORSEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:25: Cys Ala Gly Ala Thr Gly Thr Ala Cys Cys GlyThr Cys Thr Thr Gly 1 5 10 15 Cys Ala Cys Ala Cys Gly Ala Ala 20 (2)INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Cys Ala Thr CysThr Gly Cys Thr Cys Ala Thr Cys Thr Ala Cys Cys 1 5 10 15 Thr Cys AlaCys Cys Ala Thr Gly 20 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:27: Cys Ala Thr Ala Gly Gly Ala Ala Ala Cys Ala Thr Ala Gly Cys Gly 15 10 15 Thr Gly Cys Gly Thr Cys Cys Gly 20 (2) INFORMATION FOR SEQ IDNO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:28: Ala Ala Gly Cys Thr Thr Cys Thr Ala Gly AlaGly Ala Thr Cys Cys 1 5 10 15 Cys Thr Cys Gly Ala Cys Cys Thr Cys 20 25(2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Ala Gly Gly CysGly Cys Ala Gly Ala Ala Cys Thr Gly Gly Thr Ala 1 5 10 15 Gly Gly ThrAla Thr Gly Gly Ala Ala 20 25 (2) INFORMATION FOR SEQ ID NO:30: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:30: Gly Cys Thr Cys Ala Thr Cys Cys Thr Cys Thr Gly Cys ThrThr Cys 1 5 10 15 Thr Gly Gly Thr Ala Cys Gly 20 (2) INFORMATION FOR SEQID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:31: Cys Ala Gly Ala Thr Gly Thr Ala Cys Cys GlyThr Cys Thr Thr Gly 1 5 10 15 Cys Ala Cys Ala Cys Gly Ala Ala 20 (2)INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:34 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Cys Gly Ala GlyGly Ala Thr Cys Cys Cys Ala Ala Cys Thr Thr Thr 1 5 10 15 Gly Cys CysThr Cys Thr Gly Cys Thr Thr Thr Thr Thr Gly Gly Thr 20 25 30 Gly Gly (2)INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Cys Cys Thr CysAla Gly Thr Gly Ala Ala Gly Gly Gly Ala Ala Thr 1 5 10 15 Gly Gly GlyAla Gly Cys Gly Ala 20 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:34: Cys Thr Thr Gly Cys Thr Thr Gly Thr Ala Cys Gly Cys Cys Thr Thr 15 10 15 Cys Cys Gly Gly Ala Ala Gly Thr 20 (2) INFORMATION FOR SEQ IDNO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:35: Thr Gly Gly Gly Cys Ala Ala Cys Ala Gly CysCys Thr Ala Gly Thr 1 5 10 15 Gly Ala Thr Cys Ala Cys Cys Gly 20 (2)INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Cys Thr Gly CysThr Cys Cys Cys Ala Gly Cys Ala Gly Ala Ala Gly 1 5 10 15 Gly Thr CysThr Gly Gly Thr Thr 20 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:37: Ala Thr Gly Ala Ala Thr Gly Gly Cys Thr Cys Cys Gly Gly Cys Ala 15 10 15 Gly Cys Cys Ala Gly Gly Gly 20 (2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:38: Thr Thr Gly Gly Ala Gly Ala Cys Cys Ala Gly Ala Gly CysGly Thr 1 5 10 15 Ala Ala Ala Cys Gly Ala Thr Gly Gly 20 25 (2)INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Ala Gly Ala ThrGly Gly Cys Thr Gly Ala Cys Ala Thr Cys Cys Ala 1 5 10 15 Gly Ala AlaCys Ala Thr Thr Thr Cys Gly Cys Thr Gly Gly Ala Cys 20 25 30 Ala Gly CysCys Cys Ala Gly Gly Gly Ala Gly Cys Gly 35 40 45 (2) INFORMATION FOR SEQID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:40: Ala Thr Cys Ala Cys Ala Gly Gly Cys Ala CysThr Gly Cys Cys Ala 1 5 10 15 Cys Ala Gly Cys Cys Cys Cys Thr Ala CysGly Cys Thr Cys Cys Cys 20 25 30 Thr Gly Gly Gly Cys Thr Gly Thr Cys CysAla Gly Cys Gly 35 40 45 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:41: Ala Thr Gly Gly Cys Thr Gly Ala Thr Gly Cys Cys Cys Ala Gly Ala 15 10 15 Ala Cys Ala Thr Thr Thr Cys Ala Cys 20 25 (2) INFORMATION FORSEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:42: Ala Gly Cys Cys Ala Gly Gly Cys Ala Thr CysCys Ala Gly Cys Gly 1 5 10 15 Thr Gly Thr Ala Gly Ala Thr 20 (2)INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Ala Cys Gly GlyThr Cys Gly Cys Thr Thr Cys Gly Cys Cys Thr Thr 1 5 10 15 Cys Ala GlyCys Cys Cys Gly Gly Cys Cys Ala Cys Cys Thr Ala Cys 20 25 30 Gly Cys CysThr Gly Thr Cys Gly Cys Cys Thr Gly Gly 35 40 45 (2) INFORMATION FOR SEQID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:44: Ala Cys Gly Gly Thr Cys Gly Cys Thr Thr CysGly Cys Cys Thr Thr 1 5 10 15 Cys Ala Gly Cys Cys Cys Gly Gly Cys CysAla Cys Cys Thr Ala Cys 20 25 30 Gly Cys Cys Thr Gly Thr Cys Gly Cys CysThr Gly Gly 35 40 45 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:45: Gly Cys Gly Cys Ala Ala Cys Gly Cys Gly Cys Gly Cys Gly Cys Cys 15 10 15 Gly Cys Cys Gly Gly Gly Gly Gly Gly Cys Thr Cys Gly Thr Gly Thr20 25 30 Gly Gly Cys Thr Gly Cys Thr Gly Gly Cys Gly Gly Cys 35 40 45(2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Ala Thr Cys ThrAla Cys Ala Cys Gly Cys Gly Gly Gly Ala Thr Gly 1 5 10 15 Cys Cys ThrGly Gly Cys Thr Cys Thr Thr Thr Gly Gly Gly Gly Cys 20 25 30 Cys Cys ThrCys Gly Thr Cys Thr Gly Cys Ala Ala Gly 35 40 45 (2) INFORMATION FOR SEQID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:47: Ala Thr Cys Thr Ala Cys Ala Cys Gly Cys ThrGly Gly Ala Thr Gly 1 5 10 15 Cys Cys Cys Thr Gly Gly Cys Thr 20 (2)INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: Cys Gly Thr AlaGly Cys Gly Cys Ala Cys Gly Gly Thr Gly Cys Cys 1 5 10 15 Gly Thr AlaGly Thr Ala 20 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 40 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:49: Gly Ala Thr Gly Gly Ala Thr Cys Cys Gly Cys Cys Ala Cys Cys Ala 15 10 15 Thr Gly Gly Cys Thr Gly Ala Thr Gly Cys Cys Cys Ala Gly Ala Ala20 25 30 Cys Ala Thr Thr Thr Cys Ala Cys 35 40 (2) INFORMATION FOR SEQID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:50: Gly Cys Ala Gly Gly Thr Ala Cys Cys Thr GlyThr Cys Cys Ala Cys 1 5 10 15 Gly Gly Ala Gly Ala Cys Ala Gly Cys AlaGly Cys 20 25 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:51: Gly Ala Thr Gly Gly Cys Thr Gly Ala Thr Gly Cys Cys Cys Ala Gly 15 10 15 Ala Ala Cys Ala Thr Thr Thr Cys Ala Cys Thr Gly Gly Ala Cys Ala20 25 30 Gly Cys Cys Cys Ala Gly Gly Gly Ala Gly Thr Gly Thr 35 40 45(2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv)ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: Gly Ala Cys CysAla Cys Ala Gly Gly Cys Ala Cys Thr Gly Cys Cys 1 5 10 15 Ala Cys GlyGly Cys Cys Cys Cys Cys Ala Cys Ala Cys Thr Cys Cys 20 25 30 Cys Thr GlyGly Gly Cys Thr Gly Thr Cys Cys Ala Gly 35 40 45 (2) INFORMATION FOR SEQID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:53: Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly CysCys Cys Ala Ala Gly 1 5 10 15 Thr Gly Cys Cys Thr Gly Gly Cys Ala GlyGly Ala Gly Cys Cys Ala 20 25 30 Ala Gly Cys Ala Gly Thr Ala Cys Cys AlaCys Ala Gly 35 40 45 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 37 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:54: Cys Gly Cys Gly Gly Ala Thr Cys Cys Ala Thr Thr Ala Thr Gly Thr 15 10 15 Cys Thr Gly Cys Ala Cys Thr Cys Cys Gly Ala Ala Gly Gly Ala Ala20 25 30 Ala Thr Thr Thr Gly 35 (2) INFORMATION FOR SEQ ID NO:55: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:SEQ ID NO:55: Cys Gly Cys Gly Ala Ala Thr Thr Cys Thr Thr Ala Thr GlyThr Gly 1 5 10 15 Ala Ala Gly Cys Gly Ala Thr Cys Ala Gly Ala Gly ThrThr Cys Ala 20 25 30 Thr Thr Thr Thr Thr Cys 35 (2) INFORMATION FOR SEQID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:56: Gly Cys Gly Gly Gly Ala Thr Cys Cys Gly CysThr Ala Thr Gly Gly 1 5 10 15 Cys Thr Gly Gly Thr Gly Ala Thr Thr CysThr Ala Gly Gly Ala Ala 20 25 30 Thr Gly (2) INFORMATION FOR SEQ IDNO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCEDESCRIPTION: SEQ ID NO:57: Cys Cys Gly Gly Ala Ala Thr Thr Cys Cys CysCys Thr Cys Ala Cys 1 5 10 15 Ala Cys Cys Gly Ala Gly Cys Cys Cys CysThr Gly Gly 20 25 (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 50 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:58: Cys Cys Ala Ala Gly Cys Thr Thr Cys Thr Ala Ala Thr Ala Cys Gly 15 10 15 Ala Cys Thr Cys Ala Cys Thr Ala Thr Ala Gly Gly Gly Cys Cys Ala20 25 30 Cys Cys Ala Thr Gly Gly Cys Thr Gly Ala Thr Gly Cys Cys Cys Ala35 40 45 Gly Ala 50 (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 57 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ IDNO:59: Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr 15 10 15 Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr20 25 30 Thr Thr Thr Gly Cys Ala Gly Gly Gly Thr Thr Thr Ala Thr Thr Cys35 40 45 Cys Gly Gly Thr Cys Cys Thr Cys Gly 50 55

What is claimed is:
 1. An isolated nucleic acid encoding a human or ratgalanin receptor (GALR3), wherein the human galanin receptor (GALR3) hasan amino acid sequence identical to the amino acid sequence shown inFIG. 4 (SEQ ID NO: 4) or that encoded by plasmid pEXJ-hGalR3 (ATCCAccession No. 97827), and the rat galanin receptor (GALR3) has an aminoacid sequence identical t the amino acid sequence shown in FIG. 2 (SEQID NO: 2) or that encoded by plasmid pEXJ-rGalR3T (ATCC Accession No.97826) or plasmid K1086 (ATCC Accession No. 97747).
 2. The nucleic acidof claim 1, wherein the nucleic acid is DNA.
 3. The nucleic acid ofclaim 2, wherein the DNA is cDNA.
 4. The nucleic acid of claim 1,wherein the nucleic acid is RNA.
 5. The nucleic acid of claim 1, whereinthe nucleic acid encodes a rat galanin receptor (GALR3).
 6. The nucleicacid of claim 1, wherein the nucleic acid encodes a human galaninreceptor (GALR3).
 7. The nucleic acid of claim 5, wherein the nucleicacid encodes a rat galanin receptor (GALR3) which has an amino acidsequence encoded by the plasmid K1086 (ATCC Accession No. 97747).
 8. Thenucleic acid of claim 5, wherein the nucleic acid encodes a rat galaninreceptor (GALR3) having the amino acid sequence shown in FIG. 2 (SEQ IDNO: 2).
 9. The nucleic acid of claim 6, wherein the nucleic acid encodesa human galanin receptor (GALR3) which has an amino acid sequenceencoded by the plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
 10. Thenucleic acid of claim 6, wherein the human galanin receptor (GALR3) hasa sequence, which sequence comprises the sequence shown in FIG. 4 (SEQID NO: 4) from amino acid 60 through amino acid
 427. 11. An isolatedvector consisting essentially of the nucleic acid of claim
 1. 12. Anisolated vector consisting essentially of the nucleic acid of claim 6.13. The vector of claim 11 adapted for expression in a bacterial cellwhich comprises the regulatory elements necessary for expression of thenucleic acid in the bacterial cell operatively linked to the nucleicacid encoding a galanin receptor (GALR3) as to permit expressionthereof.
 14. The vector of claim 11 adapted for expression in anamphibian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the amphibian cell operatively linkedto the nucleic acid encoding a galanin receptor (GALR3) as to permitexpression thereof.
 15. The vector of claim 11 adapted for expression ina yeast cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the yeast cell operatively linked tothe nucleic acid encoding a GALR3 receptor as to permit expressionthereof.
 16. The vector of claim 11 adapted for expression in an insectcell which comprises the regulatory elements necessary for expression ofthe nucleic acid in the insect cell operatively linked to the nucleicacid encoding the galanin receptor (GALR3) as to permit expressionthereof.
 17. The isolated vector of claim 16 which comprises abaculovirus.
 18. The vector of claim 11 adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding a galanin receptor (GALR3) as to permitexpression thereof.
 19. The vector of claim 11 wherein the vector is aplasmid.
 20. The vector of claim 19 wherein the plasmid is designatedK1086 (ATCC Accession No. 97747).
 21. The vector of claim 19 wherein theplasmid is designated pEXJ-hGalR3 (ATCC Accession No. 97827).
 22. Thevector of claim 19 wherein the plasmid is designated pEXJ-RGalR3T (ATCCAccession No. 97826).
 23. A cell comprising the vector of claim
 11. 24.The cell of claim 23, wherein the cell is a non-mammalian cell.
 25. Thecell of claim 24, wherein the non-mammalian cell is a Xenopus oocytecell or a Xenopus melanophore cell.
 26. The cell of claim 23, whereinthe cell is a mammalian cell.
 27. The cell of claim 26, wherein the cellis a COS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, amouse Y1 cell, a LM(tk-) cell or a CHO cell.
 28. The cell of claim 27designated 293-rGalR3-105 (ATCC Accession No. CRL-12287).
 29. The cellof claim 27 designated L-hGalR3-228 (ATCC Accession No. CRL-12373). 30.An insect cell comprising the vector of claim
 16. 31. The insect cell ofclaim 30, wherein the insect cell is an Sf9 cell.
 32. The insect cell ofclaim 30, wherein the insect cell is an Sf21 cell.
 33. A membranepreparation isolated from the cell of claim 23 or 30.